Compositions and methods using methanotrophic s-layer proteins for expression of heterologous proteins

ABSTRACT

In alternative embodiments, provided are compositions and methods for making a chimeric polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide. In alternative embodiments, the compositions and methods comprise recombinantly engineering a methylotrophic or methanotrophic bacteria to recombinantly express a chimeric polypeptide comprising an S-layer polypeptide and a heterologous polypeptide or peptide. Also provided are compositions and methods for displaying or immobilizing proteins on a methanotrophic S-layer. In alternative embodiments, provided are compositions and methods comprising recombinant methylotrophic or methanotrophic bacteria comprising assembled or self-assembled recombinant or isolated chimeric S-layer polypeptides. In alternative embodiments, provided are compositions and methods using recombinant methylotrophic or methanotrophic bacteria, optionally a Methylomicrobium alcaliphilum, optionally a M. alcaliphilum sp. 20Z, for ectoine ((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), for the production or synthesis of a protein, e.g., an ectoine, or an enzyme, e.g., a lipase.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. (U.S. Ser. No.) 62/551,502, filed Aug. 29,2017, and U.S. Ser. No. 62/551,490, filed Aug. 29, 2017. Theaforementioned applications are expressly incorporated herein byreference in their entirety and for all purposes.

TECHNICAL FIELD

This invention generally relates to microbiology and bioengineering. Inalternative embodiments, provided are compositions and methods formaking a chimeric polypeptide comprising an S-layer polypeptide and aheterologous polypeptide or peptide. In alternative embodiments, thecompositions and methods comprise recombinantly engineering amethylotrophic or methanotrophic bacteria to recombinantly express achimeric polypeptide comprising an S-layer polypeptide and aheterologous polypeptide or peptide. Also provided are compositions andmethods for displaying or immobilizing proteins on a methanotrophicS-layer. In alternative embodiments, provided are compositions andmethods using recombinant methylotrophic or methanotrophic bacteria,optionally a Methylomicrobium alcaliphilum (M. alcaliphilum), optionallya M. alcaliphilum sp. 20Z, for ectoine((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), for theproduction or synthesis of a protein, e.g., an ectoine, or an enzyme,e.g., a lipase.

BACKGROUND

Bacterial cell surface layers are regular para-crystalline structuresthat cover the entire surface of a cell and consist of a single layer ofidentical proteins or glycoproteins. These glycoproteins are potentiallyof industrial interest because they intrinsically self-assemble andre-crystallise to form porous semi-permeable membranes. Thesecharacteristics, and subsequent functionalisation of surfaces, has ledto new types of ultrafiltration membranes, affinity structures, enzymemembranes, micro-carriers, biosensors, diagnostic devices, biocompatiblesurfaces and vaccines, as well as targeting, delivery, andencapsulation.

Methylotrophic and methanotrophic bacteria have been used as systems forthe heterologous expression of proteins, see e.g., US 2010 0221813 A1(2010). However, most attempts to improve protein expression have beenfocused on intracellular protein expression.

An S-layer, or surface layer, a part of a cell envelope found in almostall archaea and in many types of bacteria, consists of a monomolecularlayer composed of identical proteins or glycoproteins. For manybacteria, the S-layer represents the outermost interaction zone withtheir respective environments, and it can have many different functionsdepending on the species, for example an S-layer can have a mechanicaland osmotic stabilization function, can protect against bacteriophagesor phagocytosis, can provide resistance against low pH, can act as abarrier against high-molecular-weight substances, can act as a molecularsieve and barrier, can have anti-fouling properties, be involved inbiomineralization, and the like.

S-Layers are present at the surfaces of methylotrophic andmethanotrophic cells such as Methylococcus, Methylothermus, andMethylomicrobium bacterial cells. For example, differentMethylomicrobium species can synthesize S-layers with planar (p2, p4)symmetry or form cup-shaped or conical structures with hexagonal (p6)symmetry. S-layers are a well-recognized microbial product with verybroad biotechnological applications. Numerous research activities arefocused on the construction of fusion proteins (S-layer proteins withattached enzymes) for production of immobilized biocatalysts. Formationof S-layers has been observed in all tested Methylomicrobium species. M.album BG8, M. alcaliphilum 20Z and M. buryatense form S-layersconsisting of cup-shaped subunits arranged in p6 symmetry.Methanotrophic S-layers have been mentioned as a potential value-addedproduct, but were not explored much due to the lack of knowledge on itsgenetic elements.

The use of an aerobic methane-oxidation process for methane reduction incoal mines has been actively discussed for decades and even tested inthe 1980s. The approach was very simple: different methanotrophiccultures were sprayed on coal mine surfaces and methane consumption wasmonitored. The study indicated a potential for the methanotroph-basedtechnology, however, no active “industrial strain” was identified and noprofitable process was developed.

SUMMARY

In alternative embodiments, provided are methods for making a chimericpolypeptide comprising an S-layer polypeptide, or self-assembling orself-aggregating fragments thereof, and a heterologous polypeptide orpeptide, the method comprising recombinantly engineering amethylotrophic or methanotrophic bacteria to recombinantly express achimeric polypeptide comprising an S-layer polypeptide or aself-assembling fragment thereof and a heterologous polypeptide orpeptide,

-   -   wherein optionally the recombinant or isolated chimeric        polypeptide or self-assembling or self-aggregating fragment        thereof has assembled or is self-assembled to form a        monomolecular layer, and optionally the S-layer polypeptide or        self-assembling fragment thereof is on the carboxy terminal end        of the heterologous polypeptide or peptide, and optionally the        S-layer polypeptide or self-assembling fragment thereof        comprises an S-layer polypeptide endogenous to the        methylotrophic or methanotrophic bacteria, or comprises an        S-layer polypeptide from another methylotrophic or        methanotrophic bacteria or from another bacteria,

In alternative embodiments, provided are methods for displaying orimmobilizing proteins on a methanotrophic S-layer comprisingrecombinantly engineering a methylotrophic or methanotrophic bacteria torecombinantly express a chimeric polypeptide comprising an S-layerpolypeptide or a self-assembling fragment thereof and a heterologouspolypeptide or peptide,

-   -   wherein optionally the recombinant or isolated chimeric S-layer        polypeptide (or self-assembling or self-aggregating fragment        thereof) has assembled or is self-assembled to form a        monomolecular layer, and optionally the S-layer polypeptide or        self-assembling fragment thereof is on the carboxy terminal end        of the heterologous polypeptide or peptide, and optionally the        S-layer polypeptide or self-assembling fragment thereof        comprises an S-layer polypeptide endogenous to the        methylotrophic or methanotrophic bacteria, or comprises an        S-layer polypeptide or self-assembling or self-aggregating        fragment thereof from another methylotrophic or methanotrophic        bacteria or from another bacteria.

In alternative embodiments, provided are recombinant or isolatedchimeric S-layer polypeptides, wherein the recombinant or isolatedchimeric S-layer polypeptide comprises an S-layer polypeptide orself-assembling or self-aggregating fragment thereof and a heterologouspolypeptide or peptide,

-   -   wherein optionally the recombinant or isolated chimeric S-layer        polypeptide has assembled or is self-assembled to form a        monomolecular layer, and optionally the S-layer polypeptide or        self-assembling or self-aggregating fragment thereof is on the        carboxy terminal end of the heterologous polypeptide or peptide,        and optionally the S-layer polypeptide or self-assembling or        self-aggregating fragment thereof comprises an S-layer        polypeptide endogenous to the methylotrophic or methanotrophic        bacteria, or comprises an S-layer polypeptide or self-assembling        or self-aggregating fragment thereof from another methylotrophic        or methanotrophic bacteria or from another bacteria.

In alternative embodiments, provided are recombinant or isolatedmonomolecular layers comprising a plurality of chimeric S-layerpolypeptides, wherein the plurality of recombinant or isolated chimericS-layer polypeptides comprise an S-layer polypeptide or self-assemblingfragment thereof and a heterologous polypeptide or peptide,

-   -   wherein optionally the plurality of recombinant or isolated        chimeric S-layer polypeptide has assembled or is self-assembled        to form a monomolecular layer, and optionally the S-layer        polypeptide or self-assembling or self-aggregating fragment        thereof is on the carboxy terminal end of the heterologous        polypeptide or peptide, and optionally the S-layer polypeptide        comprises an S-layer polypeptide or self-assembling or        self-aggregating fragment thereof endogenous to the        methylotrophic or methanotrophic bacteria, or comprises an        S-layer polypeptide or self-assembling or self-aggregating        fragment thereof from another methylotrophic or methanotrophic        bacteria or from another bacteria.

In alternative embodiments, provided are engineered or recombinantmethylotrophic or methanotrophic bacteria comprising the recombinant orisolated chimeric S-layer polypeptide as provided herein, or comprisinga recombinant or chimeric polypeptide made by the method as providedherein, wherein optionally the S-layer polypeptide or self-assembling orself-aggregating fragment thereof comprises an S-layer polypeptideendogenous to the methylotrophic or methanotrophic bacteria, orcomprises an S-layer polypeptide or self-assembling or self-aggregatingfragment thereof from another methylotrophic or methanotrophic bacteriaor from another bacteria.

In alternative embodiments, provided are recombinant or chimericpolypeptides assembled or self-assembled to form a monomolecular layeron the extracellular surface of the recombinant methylotrophic ormethanotrophic bacteria, and optionally the heterologous polypeptide orpeptide is at least partially exposed, or is fully exposed, to anextracellular environment or milieu, and optionally the S-layerpolypeptide or self-assembling or self-aggregating fragment thereof ison the carboxy terminal end of the heterologous polypeptide or peptide.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the S-layerpolypeptide or self-assembling or self-aggregating fragment thereof (orthe finally post-translationally processed S-layer polypeptide)comprises or is a lipoprotein, and optionally the S-layer polypeptidecomprises an S-layer polypeptide endogenous to the methylotrophic ormethanotrophic bacteria, or comprises an S-layer polypeptide fromanother methylotrophic or methanotrophic bacteria or from anotherbacteria.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the methylotrophic ormethanotrophic bacteria is selected the group consisting of aMethylococcus, a Methylomonas, a Methylomicrobium, a Methylobacter, aMethylomarinum, a Methylovulum, a Methylocaldum, a Methylothermus, aMethylomarinovum, a Methylosphaera, a Methylocystis, and a Methylosinusbacteria, for example, in alternative embodiments the S-layerpolypeptide is derived from a Methylococcus, a Methylomonas, aMethylomicrobium, a Methylobacter, a Methylomarinum, a Methylovulum, aMethylocaldum, a Methylothermus, a Methylomarinovum, a Methylosphaera, aMethylocystis, and a Methylosinus bacteria.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, wherein themethylotrophic or methanotrophic bacteria is a Methylomicrobiumalcaliphilum (M. alcaliphilum), or a M. alcaliphilum sp. 20Z, forexample, in alternative embodiments the S-layer polypeptide is derivedfrom a Methylomicrobium alcaliphilum (M. alcaliphilum), or a M.alcaliphilum sp. 20Z.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the chimericpolypeptide, or the recombinant or isolated chimeric S-layerpolypeptide, is expressed on the surface of a methylotrophic ormethanotrophic bacteria, and the heterologous polypeptide, or therecombinant or isolated chimeric S-layer polypeptide, or S-layerpolypeptide or self-assembling or self-aggregating fragment thereof, isat least in part exposed to an extracellular environment or milieu.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the methanotrophicS-layer polypeptide or self-assembling or self-aggregating fragmentthereof is isolated or is derived from the methylotrophic ormethanotrophic bacteria.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the heterologouspolypeptide or peptide, or recombinant or isolated chimeric S-layerpolypeptide, comprises or further comprises: an enzyme, a structuralprotein, a fluorescent or a chemiluminescent protein, a ligand, areceptor, an antibody or antigen binding protein, or an antigen, atolerogen or an immunogen.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the enzyme is anindustrial enzyme, or the enzyme is a lipase, a protease, a nitrogenase,a hydrogenase, a monooxygenase, an amylase, an isomerase, a cellulase orhemicellulase, a laccase, an epimerase, a decarboxylase, a glucanase ora fl-glucanase, a glucosidase, a phosphorylase, a phosphatase, ahalogenase or a dehalogenase, a GlcNAc transferase, anN-acetylglucosamine, a GlcNAc transferase, a neuraminidase or sialidase,a nuclease, a peroxidase or an oxidase, or a metalloproteinase.

In alternative embodiments of the methods or the recombinant or isolatedchimeric S-layer polypeptides as provided herein, the chimeric protein,the recombinant or isolated chimeric S-layer polypeptide orself-assembling or self-aggregating fragment thereof, the recombinant orisolated monomolecular layer, or the recombinant methylotrophic ormethanotrophic bacteria, act as or are used as or used for: anultrafiltration membrane; an affinity structure; nitrogen fixation;converting carbon dioxide into methane; methane uptake or methaneoxidation; converting nitrogen gas to ammonia; a membrane of an enzymemembrane; a micro-carrier; a biosensor; a diagnostic device; abiocompatible surface; a vaccine; a device or composition for targeting,delivery and/or encapsulation; an anchor for extracellular production ofa small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical.

In alternative embodiments, provided herein are membranes or an enzymemembrane; an ultrafiltration membrane; an affinity structure; acomposition or device for nitrogen fixation; a composition or device forconverting carbon dioxide into methane; a composition or device formethane uptake or methane oxidation; a composition or device forconverting nitrogen gas to ammonia; a membrane of an enzyme membrane; amicro-carrier; a biosensor; a diagnostic device; a biocompatiblesurface; a vaccine; a device or composition for targeting, deliveryand/or encapsulation; an implant; an anchor for extracellular productionof a small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical, comprising:

-   -   a chimeric polypeptide as provided herein,    -   a recombinant or isolated chimeric S-layer polypeptide or        self-assembling or self-aggregating fragment thereof as provided        herein,    -   a recombinant or isolated monomolecular layer as provided        herein, or    -   a recombinant methylotrophic or methanotrophic bacteria as        provided herein.

In alternative embodiments, provided are recombinant or engineeredmethylotrophic or methanotrophic bacteria, optionally a Methylomicrobiumalcaliphilum (M. alcaliphilum), optionally a M. alcaliphilum sp. 20Z,for ectoine ((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylicacid) production or synthesis, wherein:

-   -   (a) the recombinant or engineered methylotrophic or        methanotrophic bacteria comprises an ectoine biosynthetic gene        cluster organized as one operon (ectABC-ask), wherein the operon        comprises genes encoding: a diaminobutyric acid (DABA)        aminotransferase (EctB); a DABA acetyltransferase (EctA), and an        ectoine synthase (EctC); and    -   (b) the recombinant or engineered methylotrophic or        methanotrophic bacteria: (i) is engineered to lack or not        express a functional EctR1 repressor; (ii) comprises an        isocitrate lyase/malate synthase fusion under (transcriptionally        controlled by) a hps promoter (P_(hps)); and/or, (iii) comprises        one or more of the genetic modifications set forth in Table 1        (see Example 2, below).

In alternative embodiments of the recombinant or engineeredmethylotrophic or methanotrophic bacteria, a doeA-gene encoding ectoinehydrolase is deleted or mutated such that a functional ectoine hydrolaseis not expressed.

In alternative embodiments, the recombinant or engineered methylotrophicor methanotrophic bacteria further comprises an exogenous nucleic acidcapable of expressing a methanotrophic lipase, or a functional lipasefragment thereof (optionally a LipL1 expression plasmid), in therecombinant or engineered methylotrophic or methanotrophic bacteria.

In alternative embodiments, the recombinant or engineered bacteria isengineered such that the ectoine and/or the lipase, or the functionallipase fragment thereof, is expressed as an S layer protein chimericpolypeptide, optionally as a lipase-S protein fusion protein (an Slayer-lipase or an S layer-ectoine fusion protein).

In alternative embodiments, the methylotrophic or methanotrophicbacteria is selected the group consisting of a Methylococcus, aMethylomonas, a Methylomicrobium, a Methylobacter, a Methylomarinum, aMethylovulum, a Methylocaldum, a Methylothermus, a Methylomarinovum, aMethylosphaera, a Methylocystis, and a Methylosinus bacteria, andoptionally the S layer protein is derived from a Methylococcus, aMethylomonas, a Methylomicrobium, a Methylobacter, a Methylomarinum, aMethylovulum, a Methylocaldum, a Methylothermus, a Methylomarinovum, aMethylosphaera, a Methylocystis, or a Methylosinus bacteria, oroptionally the S-layer polypeptide is derived from a Methylomicrobiumalcaliphilum (M. alcaliphilum), or a M. alcaliphilum sp. 20Z. Inalternative embodiments, the S-layer protein is endogenous to themethylotrophic or methanotrophic recombinant or engineered bacteria.

In alternative embodiments, the methylotrophic or methanotrophicbacteria further comprise the ability to express a heterologous orexogenous protein or enzyme, optionally an industrial enzyme; or the Slayer protein chimeric polypeptide comprises a protein or an enzyme,optionally an industrial enzyme. In alternative embodiments, the enzymeis a lipase, a protease, a nitrogenase, a hydrogenase, a monooxygenase,an amylase, an isomerase, a cellulase or hemicellulase, a laccase, anepimerase, a decarboxylase, a glucanase or a fl-glucanase, aglucosidase, a phosphorylase, a phosphatase, a halogenase or adehalogenase, a GlcNAc transferase, an N-acetylglucosamine, a GlcNActransferase, a neuraminidase or sialidase, a nuclease, a peroxidase oran oxidase, or a metalloproteinase.

In alternative embodiments, the recombinant or engineered methylotrophicor methanotrophic bacteria, or the S layer protein chimeric polypeptideproduced by the recombinant or engineered methylotrophic ormethanotrophic bacteria, act as or are used as or used for: anultrafiltration membrane; an affinity structure; nitrogen fixation;converting carbon dioxide into methane; methane uptake or methaneoxidation; converting nitrogen gas to ammonia; a membrane of an enzymemembrane; a micro-carrier; a biosensor; a diagnostic device; abiocompatible surface; a vaccine; a device or composition for targeting,delivery and/or encapsulation; an anchor for extracellular production ofa small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical.

In alternative embodiments, provided are: a membrane or an enzymemembrane; an ultrafiltration membrane; an affinity structure; acomposition or device for nitrogen fixation; a composition or device forconverting carbon dioxide into methane; a composition or device formethane uptake or methane oxidation; a composition or device forconverting nitrogen gas to ammonia; a membrane of an enzyme membrane; amicro-carrier; a biosensor; a diagnostic device; a biocompatiblesurface; a vaccine; a device or composition for targeting, deliveryand/or encapsulation; an implant; an anchor for extracellular productionof a small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical, comprising:

-   -   a chimeric polypeptide made by a method (or by a recombinant or        engineered methylotrophic or methanotrophic bacteria) as        provided herein,    -   a recombinant or isolated chimeric S-layer polypeptide made by a        method (or by a recombinant or engineered methylotrophic or        methanotrophic bacteria) as provided herein,    -   a recombinant or isolated monomolecular layer made by a method        (or by a recombinant or engineered methylotrophic or        methanotrophic bacteria) as provided herein, or    -   a recombinant or engineered methylotrophic or methanotrophic        bacteria made by a method as provided herein.

The details of one or more embodiments as provided herein are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments asprovided herein and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1A-B illustrate Lipase production in methanotrophic cultures:

FIG. 1A illustrates an image of in vivo activity, or intracellularproduction, of a lipase gene cloned into an expression vector andintroduced into Methylomicrobium sp. AP 18, as detected on rhodamineB-containing plates;

FIG. 1B illustrates an image of a Coumassie-stained polyacrylamide gel(PAAG) showing that a lipase gene expression vector showed low levels oflipase expression, e.g., the protein was not visible in lane 5; and anoptimized vector with a ribosome binding site (RBS) resulted in cloneswith significantly higher expression, with the recombinant lipasecomprising of about 1% to 2% of total cell protein, see lanes 6 to 12,

as described in detail in Example 1, below.

FIG. 2A-B illustrate construction and expression of a vector containinga novel genetically altered microbial catalyst producing lipase:

FIG. 2A, schematically illustrates in upper and lower images a lipaseenzyme-encoding nucleic acid construct, and a plasmid containing thisgenetic construct, respectively;

FIG. 2B schematically illustrates how this genetic construct istransferred from E. coli S17-1 by conjugation and plasmid transfer intoan M. alcaliphilum 20Z strain, followed by recombination andincorporation of the fused gene (the genetic construct) into thechromosome, resulting in expression and export of the fusion protein, asdescribed in detail in Example 1, below.

FIG. 3 , schematically illustrates the ectoine biosynthesis pathway inM. alcaliphilum 20Z, including three specific enzymes: diaminobutyricacid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), andectoine synthase (EctC), as described in detail in Example 2, below.

FIG. 4A-C: FIG. 4A graphically illustrates data showing the growth of M.alcaliphilum 20ZR in a DASBOX™ mini bioreactor, as batch and chemostatmode; FIG. 4B-C graphically illustrate O₂ and CH₄ consumptions and CO₂production in steady-state for bioreactor replicate 1 (FIG. 4B) andbioreactor replicate 2 (FIG. 4C), as described in detail in Example 2,below.

FIG. 5A-B: FIG. 5A illustrates a chromatogram of 1 mM ectoine solution,and FIG. 5B illustrates a chromatogram of 20ZR cell extract, asdescribed in detail in Example 2, below.

FIG. 6 illustrates a chromatogram of an HPLC analysis of 20Z^(R)wild-type as a batch culture, as described in detail in Example 2,below.

FIG. 7 illustrates a chromatogram of an HPLC analysis of 20Z^(R)ΔectRstrain in batch culture, as described in detail in Example 2, below.

FIG. 8 illustrates a chromatogram of an HPLC analysis of20Z^(R)ΔectRΔdoeA strain (batch culture), as described in detail inExample 2, below.

FIG. 9A-B illustrates chromatograms HPLC analyses that reveal thehighest levels of ectoine: FIG. 9A illustrates HPLC analysis of HPLCanalysis of 20Z::P_(SL)-L1ΔectR (in batch culture); and, FIG. 9Billustrates 20Z^(R)P_(SL)-L1ΔectR ΔdoeA strain (in batch culture), asdescribed in detail in Example 2, below.

FIG. 10A-C illustrate data from the cultivation of TWC #G2-3 asperformed in a DASBOX™ mini bioreactor, and collected data is shown as:FIG. 10A graphically illustrates growth of M. alcaliphilum20ZRPsL-L1ΔectRΔdocA as batch and chemostat mode; FIG. 10B-C graphicallyillustrate O₂ and CH₄ consumptions and CO₂ production in steady-statefor bioreactor replicate 1 (FIG. 10B) and bioreactor replicate 2 (FIG.10C), as described in detail in Example 2, below.

FIG. 11 illustrates LipL1 preparations, as described in detail inExample 2, below.

FIG. 12 illustrates an image of production of the GFP-comprisingrecombinant protein by the 20ZR-L1-SL strain, as described in detail inExample 3, below.

FIG. 13 schematically illustrates exemplary genetic constructscontaining self-cleavable inteins, which were inserted between thelipase and the S-layer, as described in detail in Example 3, below.

FIG. 14 illustrates an image of extracellular lipase localization andactivity in a Rhodamine B assay with TWC #11 (top left of the plate),wild type (WT) (top right of the plate) and lipL-Ssp DnaB intein mutants(bottom of the plate), as described in detail in Example 3, below.

FIG. 15 illustrates an image of a gel separating amplified nucleic acidfrom a PCR-genotyping of TWC #G14 20ZR::SL_(Nter)-LipL1-Mxe GyrA strain:FIG. 15 left: LipL locus, indicating that LipL-MxeGyrA- was incorporatedinto the genome; FIG. 15 right: LipL-S-layer locus from showing thatLipL gene was incorporated in correct orientation, asLipL-MxeGyrA-S-layer, as described in detail in Example 3, below.

FIG. 16A-E illustrate images of cells harboring various GFP fusions:FIG. 16A-B illustrate M. alcaliphilum 20ZR wild type (FIG. 16A, phase;FIG. 16B, GFP ex450-490 nm; em 500-550 nm); FIG. 16C,20Z^(R)::GFP-300Cter_(SLP); FIG. 16D, GFP-100Cter_(SLP); FIG. 16E,GFP-12Cter_(SLP), as described in detail in Example 3, below.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION Chimeric Polypeptides Comprising an S-LayerPolypeptide, or Self-Assembling or Self-Aggregating Fragments Thereof,and a Heterologous Polypeptide or Peptide

In alternative embodiments, provided are compositions and methods formaking a chimeric polypeptide comprising an S-layer polypeptide, orself-assembling or self-aggregating fragments thereof, and aheterologous polypeptide or peptide. In alternative embodiments, thecompositions and methods comprise recombinantly engineering amethylotrophic or methanotrophic bacteria to recombinantly express achimeric polypeptide comprising an S-layer polypeptide, orself-assembling or self-aggregating fragments thereof, and aheterologous polypeptide or peptide. In alternative embodiments, theS-layer polypeptide, or self-assembling or self-aggregating fragmentsthereof, is engineered to be amino terminal to, internal to, or carboxyterminal to, the heterologous polypeptide or peptide.

Also provided are compositions and methods for displaying orimmobilizing proteins on a methanotrophic S-layer. In alternativeembodiments, provided are compositions and methods comprising arecombinant or isolated chimeric S-layer polypeptide, wherein therecombinant or isolated chimeric S-layer polypeptide comprises anS-layer polypeptide, or self-assembling or self-aggregating fragmentsthereof, and a heterologous polypeptide or peptide. In alternativeembodiments, the S-layer polypeptide, or self-assembling fragments orself-aggregating thereof, is engineered to be amino terminal to,internal to, or carboxy terminal to, the heterologous polypeptide orpeptide.

Also provided are recombinant or isolated monomolecular layerscomprising a chimeric S-layer polypeptide, where the S-layer polypeptidecan be a self-assembling or self-aggregating fragment thereof. Inalternative embodiments, provided are compositions and methodscomprising recombinant methylotrophic or methanotrophic bacteriacomprising assembled or self-assembled recombinant or isolated chimericS-layer polypeptides. In alternative embodiments, the S-layerpolypeptides, or self-assembling or self-aggregating fragments thereof,are lipoproteins, for example, the S-layer polypeptides can belipoproteins as post-translationally modified.

Provided herein for the first time are applications of bacterialextracellular methanotrophic S-layer proteins, or self-assembling orself-aggregating fragments thereof, for the expression of heterologousproteins, including extracellular expression of a heterologous proteinon the surface of a methanotrophic S-layer protein-expressing bacteria.In alternative embodiments, methanotrophic S-layers, either isolated(e.g., as described in Khmelenina V N, et al (1999) Arch. Microbiol.172: 321-329) or as surface-expressed methanotrophic S-layers, are usedas an anchor or expression vehicle for the extracellular production,expression and use of proteins, e.g., enzymes such as industrialenzymes, e.g. proteinases, lipases, amylases, celluloses, fl-glucanase,as well as for their use as enzymatic systems for bioremediation andbio-mitigations, e.g. dehalogenases and peroxidases, and protein-basedbiopharmaceuticals.

We identified the gene encoding the major S-layer protein in M.alcaliphilum sp. 20Z using quantitative proteomics on purified S-layerpreparations. The S-layer protein appears to be the main cellularprotein, comprising up to 20% of total cellular protein. Provided hereinare recombinant S-layers and uses of recombinant S-layers as anefficient chimeric polypeptide or cellular system for use as anultrafiltration membrane; an affinity structure; a membrane of an enzymemembrane; a micro-carrier, a biosensor; a diagnostic device, abiocompatible surface, a vaccine, a device or composition for targeting,delivery and/or encapsulation; an anchor for extracellular production ofa small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical

In alternative embodiments of compositions and methods as providedherein, chimeric proteins are delivered and expressed outside of thebacterial cell, e.g., chimeric proteins as provided herein are expressedextracellularly can be completely or partially exposed to anextracellular milieu. In alternative embodiments, systems as providedherein are also used to produce enzymatic or structural membranes.

We developed a protocol for genetic alterations of the S-layers forheterologous expression of proteins on a bacterial cell surface.Overview of the approach is shown in FIG. 2 .

S-Layer Polypeptides, or Self-Assembling or Self-Aggregating FragmentsThereof

Exemplary S-layer polypeptides, or self-assembling or self-aggregatingfragments thereof, can comprise or consist of:

SEQ ID NO: 8 GANNQVAALQTAAGGAFDGTFFDVLSNFTVEASFEVLSQFDPETTLFVANPIVEDVNFDIVRDVNGDVTSVSVSGGSSLGFAQSDAGFQELLEAGQVTEVVFENVGSLNSILVSGNFVGSYDAGGIFYESTFEFGANAGSVAEGVGTDGNIFTIAEFTAGAAASDILDFTAMPVDNTNTAPATGHEFIAVGTEASIGDDATIIVFTAGVAADAATIVTQFADGAGDFRSADATARNADFAIDSQLIFLIDDGAGNTGVWYWDDTVGAVGDGIVDADELSQIAQLTGVVTAELTV DNFVLA, SEQ ID NO: 9TIIVFTAGVAADAATIVTQFADGAGDFRSADATARNADFAIDSQLIFLIDDGAGNTGVWYWDDTVGAVGDGIVDADELSQIAQLTGVVTAELTVDNFV LA, SEQ ID NO: 10AGNTGVWYWDDTVGAVGDGIVDADELSQIAQLTGVVTAELTVDNFVLA, SEQ ID NO: 11VGAVGDGIVDADELSQIAQLTGVVTAELTVDNFVLA, SEQ ID NO: 12 TAELTVDNFVLA,or

-   -   the S-layer polypeptide as encoded by the nucleic acid sequence        of SEQ ID NO:5, SEQ ID NO:7 and/or SEQ ID NO:7, as indicated        below.

Exemplary S-layer polypeptide self-assembling or self-aggregatingfragments thereof also can comprise or consist of any self-assemblingfragment, which can be readily identified by routine screening of anS-layer polypeptide.

Exemplary S-layer polypeptides or self-assembling or self-aggregatingfragments thereof also comprise S-layer polypeptide sequences asdescribed herein but comprising at least one amino acid residueconservative substitution, wherein optionally the at least oneconservative substitution comprises replacement of an aliphatic aminoacid with another aliphatic amino acid; replacement of a serine with athreonine or vice versa; replacement of an acidic residue with anotheracidic residue; replacement of a residue bearing an amide group withanother residue bearing an amide group; exchange of a basic residue withanother basic residue; or, replacement of an aromatic residue withanother aromatic residue, or a combination thereof, and optionally thealiphatic residue comprises Alanine, Valine, Leucine, Isoleucine or asynthetic equivalent thereof; the acidic residue comprises Asparticacid, Glutamic acid or a synthetic equivalent thereof; the residuecomprising an amide group comprises Aspartic acid, Glutamic acid or asynthetic equivalent thereof; the basic residue comprises Lysine,Arginine or a synthetic equivalent thereof; or, the aromatic residuecomprises Phenylalanine, Tyrosine or a synthetic equivalent thereof.

Recombinant Methylotrophic or Methanotrophic Bacteria to ExpressHeterologous Proteins

In alternative embodiments, provided are compositions and methods usingrecombinant methylotrophic or methanotrophic bacteria, optionally aMethylomicrobium alcaliphilum (M. alcaliphilum), optionally a M.alcaliphilum sp. 20Z, for ectoine((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), for theproduction or synthesis of a protein, e.g., an ectoine(1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), or an enzyme,e.g., a lipase.

Provided herein are new mitigation strategies for effective conversionof atmospheric greenhouse gases (e.g., CO₂ or methane) to nextgeneration chemicals which are a new technology for thereduction/stabilization of global warming. In alternative embodiments,provided are new biological processes for efficient utilization ofmethane, e.g., coal-mine methane, and also optionally comprising thesimultaneous production of e.g., amino acids; osmo-protecting,moisturizing and hydrating agents; and, industrial and/or digestiveenzymes. In alternative embodiments, these processes provide bothenvironmental (reduction of the global warming impact) and economical(production of value-added compounds) benefits. In alternativeembodiments, provided are:

-   -   i) Recombinant or engineered obligate methane-oxidizing bacteria        (methanotrophs) and methanotrophic catalysts to enhance ectoine        production capabilities in the cells;    -   ii) novel genetically altered obligate methane-oxidizing        bacteria (methanotrophs) and methanotrophic catalysts that        produce a lipase or an ectoine; iii) uses of stranded methane        emissions, such as abandoned coal mines, to “feed” recombinant        obligate methane-oxidizing bacteria (methanotrophs) as provided        herein methane (to provide methane as a carbon source) for e.g.,        methane uptake or methane oxidation.

In alternative embodiments, methods provided herein comprise use ofbiological systems (microbial cells, including recombinant obligatemethane-oxidizing bacteria (methanotrophs), or enzymes as providedherein) as catalysts for conversion of atmospheric greenhouse gases(e.g., CO₂ or methane). In alternative embodiment, methods providedherein comprise use of obligate methane-oxidizing bacteria(methanotrophs), which are a highly-specialized group of bacteriautilizing methane (CH₄) as a sole source of carbon and energy.Methanotrophs are ubiquitously distributed in nature and play animportant role in global carbon cycling. Also, these organisms are ofgreat importance for global warming because they reduce CH₄ emissionsfrom natural ecosystems. In alternative embodiment, methods providedherein comprise use of methanotrophs, including recombinant obligatemethane-oxidizing bacteria (methanotrophs), for the commercialproduction of both bulk and fine chemicals and bioremediation ofhazardous pollutants such as halogenated methanes and trichloroethylene(TCE).

In alternative embodiment, methods provided herein comprise use ofrecombinant or engineered aerobic methanotrophic bacteria forcontrolling/monitoring methane emissions from methane-producing zonessuch as coal mining, feedlots, etc. In alternative embodiment, methodsprovided herein are a bacteria-based methane reduction technology thatcan be cost effective and can be combined with synthesis of valuablecommercial products, such as biomass, amino acids, vitamins, andalternative fuels and chemicals.

In alternative embodiments, provided are engineered biologicalprocesses, and compositions and methods for practicing same, for thereduction of the methane content in defined space, e.g., a coal mine orindustrial (e.g., factory) environment. These embodiments provideenvironmental (e.g., reduction of the global warming impact), safety andeconomical (e.g., production of value-added compounds) benefits. Inalternative embodiments, provided is a microbial catalyst for efficientutilization of coal mine methane and, optionally, also for thesimultaneous production of ectoine and/or lipase.

In alternative embodiments, compositions and methods as provided herein,including recombinant obligate methane-oxidizing bacteria(methanotrophs) as provided herein, use microbial catalysts to enhanceectoine production capabilities up to 10% cell dry weight (CDW). Inalternative embodiments, provided are methods for the construction of anovel genetically altered microbial catalyst producing lipase,optionally up to 10% of CDW. Also provided is the testing of conditionsrelevant to small scale, mobile, field applications at sites of strandedmethane emissions, such as abandoned coal mines and identification oflab-scale cultivation parameters suitable for implementation of theproposed technology on site.

Methanotrophic S-Layers and S-Layer-Based Enzyme Immobilization

In alternative embodiments, compositions and methods as provided hereinuse S-layers, which are a well-recognized microbial product with verybroad biotechnological applications, see e.g., Egelseer et al., 2009,NanoBioTechnology (Shoseyov O & Levy I, eds), pp. 55-86. Humana Press,Totowa, NJ; or Egelseer et al., The Encyclopedia of IndustrialBiotechnology: Bioprocess, Bioseparation, and Cell Technology, Vol. 7(Flickinger M C, ed.), pp. 4424-4448. John Wiley & Sons, Inc., Hoboken,NJ. In alternative embodiments, compositions and methods as providedherein comprise the construction of fusion proteins comprising S-layerproteins with attached enzymes (or other proteins) for the production ofimmobilized biocatalysts. In alternative embodiments, S-layers derivedfrom Methylomicrobium species are used, including M. album BG8, M.alcaliphilum 20Z and M. buryatense. Formation of S-layers has beenobserved in all tested Methylomicrobium species. M. album BG8, M.alcaliphilum 20Z and M. buryatense form S-layers consisting ofcup-shaped subunits arranged in p6 symmetry [Jeffries and Wilkinson1978, Khmelenina et al., 1999]. In alternative embodiments, S-layerproteins are positioned carboxy terminal to the attached protein,although they can also be internal or amino terminus positioned.

We identified the gene encoding the major S-layer protein in M.alcaliphilum sp. 20Z using quantitative proteomics on purified S-layerpreparations; see Example 2, below. The S-layer protein appears to bethe main cellular protein, comprising up to 20% of total cellularprotein. In alternative embodiments, compositions and methods asprovided herein use S-layers as an efficient cellular system to deliverproteins outside of the cell. In alternative embodiments, this system isalso used to produce biological filters or purification systems, andenzymatic membranes.

Generating and Manipulating Nucleic Acids

In alternative embodiments, nucleic acids used to practice methods asprovided herein, or to make compositions or recombinant bacteria asprovided herein, are made, isolated and/or manipulated by, e.g., cloningand expression of cDNA libraries, amplification of message or genomicDNA by PCR, and the like. The nucleic acids and genes used to practicethis invention, including DNA, RNA, IRNA, antisense nucleic acid, cDNA,genomic DNA, vectors, viruses or hybrids thereof, can be isolated from avariety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from thesenucleic acids can be individually isolated or cloned and tested for adesired activity. Any recombinant expression system or gene therapydelivery vehicle can be used, including e.g., viral (e.g., AAVconstructs or hybrids) bacterial, fungal, mammalian, yeast, insect orplant cell expression systems or expression vehicles.

Alternatively, nucleic acids used to practice methods as providedherein, or to make compositions or recombinant bacteria as providedherein, can be synthesized in vitro by well-known chemical synthesistechniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc.105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995)Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth.Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No.4,458,066.

Techniques for the manipulation of nucleic acids used to practicemethods as provided herein, or to make compositions or recombinantbacteria as provided herein, such as, e.g., subcloning, labeling probes(e.g., random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrook,ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, ColdSpring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice methods as provided herein, or to make compositions orrecombinant bacteria as provided herein, is to clone from genomicsamples, and, if desired, screen and re-clone inserts isolated oramplified from, e.g., genomic clones or cDNA clones. Sources of nucleicacid used in the methods of the invention include genomic or cDNAlibraries contained in, e.g., mammalian artificial chromosomes (MACs),see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificialchromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeastartificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316;P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques23:120-124; cosmids, recombinant viruses, phages or plasmids.

In alternative embodiments, a heterologous peptide or polypeptide joinedor fused to a protein made by a method or a recombinant bacteria asprovided herein can be an N-terminal identification peptide whichimparts a desired characteristic, such as fluorescent detection,increased stability and/or simplified purification. Peptides andpolypeptides made by a method or a recombinant bacteria as providedherein can also be synthesized and expressed as fusion proteins with oneor more additional domains linked thereto for, e.g., producing a moreimmunogenic peptide, to more readily isolate a recombinantly synthesizedpeptide, to identify and isolate antibodies and antibody-expressing Bcells, and the like. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle WA). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego CA) between a purification domain and themotif-comprising peptide or polypeptide to facilitate purification. Forexample, an expression vector can include an epitope-encoding nucleicacid sequence linked to six histidine residues followed by a thioredoxinand an enterokinase cleavage site (see e.g., Williams (1995)Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif.12:404-414). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Nucleic acids or nucleic acid sequences used to practice embodiments asprovided herein can be an oligonucleotide, nucleotide, polynucleotide,or to a fragment of any of these, to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representa sense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin. Compoundsuse to practice this invention include “nucleic acids” or “nucleic acidsequences” including oligonucleotide, nucleotide, polynucleotide, or anyfragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA,tRNA, iRNA) of genomic or synthetic origin which may be single-strandedor double-stranded; and can be a sense or antisense strand, or a peptidenucleic acid (PNA), or any DNA-like or RNA-like material, natural orsynthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g.,e.g., double stranded iRNAs, e.g., iRNPs). Nucleic acids or nucleic acidsequences used to practice embodiments as provided herein includenucleic acids or oligonucleotides containing known analogues of naturalnucleotides. Nucleic acids or nucleic acid sequences used to practiceembodiments as provided herein include nucleic-acid-like structures withsynthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol.144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156. Nucleic acids ornucleic acid sequences used to practice embodiments as provided hereininclude “oligonucleotides” including a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandsthat may be chemically synthesized. Compounds use to practice thisinvention include synthetic oligonucleotides having no 5′ phosphate, andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

In alternative aspects, methods and recombinant bacteria as providedherein comprise use of “expression cassettes” comprising a nucleotidesequences capable of affecting expression of the nucleic acid, e.g., astructural gene or a transcript (e.g., encoding an S-layer protein,and/or an enzyme such as a lipase or a ectoine) in a host compatiblewith such sequences, such as e.g., methylotrophic and methanotrophiccells such as Methylococcus, Methylothermus, and Methylomicrobiumbacterial cells. Expression cassettes can include at least a promoteroperably linked with the polypeptide coding sequence or inhibitorysequence; and, in one aspect, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers.

In alternative aspects, expression cassettes used to practiceembodiments as provided herein also include plasmids, expressionvectors, recombinant viruses, any form of recombinant “naked DNA”vector, and the like. In alternative aspects, a “vector” used topractice embodiments as provided herein can comprise a nucleic acid thatcan infect, transfect, transiently or permanently transduce a cell. Inalternative aspects, a vector used to practice embodiments as providedherein can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. In alternative aspects, vectors used to practiceembodiments as provided herein can comprise viral or bacterial nucleicacids and/or proteins, and/or membranes (e.g., a cell membrane, a virallipid envelope, etc.). In alternative aspects, vectors used to practiceembodiments as provided herein can include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and can include both the expression and non-expressionplasmids. In alternative aspects, the vector used to practiceembodiments as provided herein can be stably replicated by the cellsduring mitosis as an autonomous structure, or can be incorporated withinthe host's genome.

In alternative aspects, “promoters” used to practice this inventioninclude all sequences capable of driving transcription of a codingsequence in a bacterial cell, e.g., a methylotrophic or methanotrophicbacterial cell. Thus, promoters used in the constructs of the inventioninclude cis-acting transcriptional control elements and regulatorysequences that are involved in regulating or modulating the timingand/or rate of transcription of a gene. For example, a promoter used topractice this invention can be a cis-acting transcriptional controlelement, including an enhancer, a promoter, a transcription terminator,an origin of replication, a chromosomal integration sequence, 5′ and 3′untranslated regions, or an intronic sequence, which are involved intranscriptional regulation. These cis-acting sequences typicallyinteract with proteins or other biomolecules to carry out (turn on/off,regulate, modulate, etc.) transcription.

Bacterial Growth Conditions

Any set of known growth conditions can be used to practice embodimentsas provided herein, for example, as described in US 2016-0237398 A1, orWO/2015/058179; exemplary growth conditions and parameters are describedin Example 1 and Example 2, below. Any known growth conditions forculturing methylotrophic and methanotrophic cells such as Methylococcus,Methylothermus, and Methylomicrobium bacterial cells can be used.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Exemplary Methods and Compositions

This example provides exemplary methods for making compositions andbacterial cells as provided herein.

Methanotrophic Strain Best Suited for Biotechnological Exploration.

Two methanotrophic cultures were established as the most promisingindustrial strains: Methylomicrobium alcaliphilum sp. 20Z andMethylomicrobium buryatenses 5G (see, e.g., Ojala et al., 2011;Kalyuzhnaya et al., 2015; Puri et al., 2015; Strong et al., 2016). WhileM. buryatenses 5G represents a fast-growing methanotroph (Td-3h), M.alcaliphilum sp. 20Z was found to be more stable at high-cell density.Furthermore, the latter strain has a greater potential for accumulationof extractable products (ectoine, glutamate, sucrose). Based on thosecharacteristics, M. alcaliphilum sp. 20Z was selected.

Genomes of both M. alcaliphilum sp. 20Z and M. buryatenses 5G weresequenced (see e.g., Vuilleumier et al., 2012). Genetic tools forefficient metabolic engineering of the strains were developed oroptimized (see e.g., Ojala et al., 2011; Puri 2015; Henard et al.,2016). The current toolbox includes: vectors for gene knockouts(incorporated via bi-parental mating or electroporation); vectors forheterologous expression with low, intermediate and high levels ofexpression; and vectors with tunable promoters. Provided is awhole-genome reconstruction of the M. alcaliphilum sp. 20Z metabolicnetwork, which is refined via metabolomics on cells grown in liquidculture, providing a computation framework for additional optimizationof metabolic pathways in producing traits.

Methanotrophic S-Layers an S-Layer-Based Enzyme Immobilization

Here we describe use of S-layers as an efficient cellular system todeliver proteins outside of the cell. In alternative embodiments, thissystem is also used to produce biological filters and enzymaticmembranes, or purification systems.

We identified the gene encoding the major S-layer protein in M.alcaliphilum sp. 20Z using quantitative proteomics on purified S-layerpreparations. The S-layer protein appears to be the main cellularprotein, comprising up to 20% of total cellular protein.

Lipase Production in Methanotrophic Cultures.

Lipase production by Bacillus stearothermophilus L1 [Kim et al. 2000] isoptimal at 60° C. to 65° C. and pH 9 to pH 11 [Kim et al. 1998]. Thislipase has been shown to have a 2 to 4 times higher activity forsaturated fatty acids compared to monounsaturated ones. This makes L1lipase a good candidate for hydrolysis of solid lipids like beef tallowand palm oil which are known to be difficult targets for currently usedlipases. L1 lipase gene was codon optimized for efficient expression inmethanotrophic host. The gene was synthetized and cloned into anexpression vector and introduced into Methylomicrobium sp. AP 18 forintracellular production; it's in vivo activity was detected onrhodamine B-containing plates (FIG. 1A). However, this construct showedlow levels of lipase expression, i.e., the protein was not visible onCoumassie-stained polyacrylamide gel (PAAG) (FIG. 1B, lane 5).

In order to increase the expression, optimization of its ribosomebinding site using in-house protocol was performed resulting inselection of clones with significantly higher expression (L1 comprisingof about 1% to 2% of total cell protein (FIG. 1B, lanes 6 to 12)).

Construction of a Novel Genetically Altered Microbial Catalyst ProducingLipase (up to 10% of CDW)

In order to further increase lipase production and simultaneouslysimplify its purification, an M. alcaliphilum 20Z strain expressing L1lipase extracellularly as a fusion with S-layer protein is constructed.The fusion protein is introduced into the M. alcaliphilum 20Z chromosomeusing its native genetic elements to ensure high expression and properextracellular localization of the fusion.

To facilitate lipase isolation, a site for HRV 3C protease is introducedbetween the S-layer and lipase polypeptides allowing the fusion proteinto be cleaved with HRV 3C protease to release functional L1 lipase intosolution, a genetic construct encoding this fusion protein isschematically illustrated in FIG. 2A, upper and lower images.

A plasmid containing this genetic construct is transferred from E. coliS17-1 by conjugation and plasmid transfer into an M. alcaliphilum 20Zstrain, followed by recombination and incorporation of the fused gene(the genetic construct) into the chromosome, resulting in expression andexport of the fusion protein, as schematically illustrated in FIG. 2B.

Methods.

The genetic manipulation includes the following set of steps:

-   -   1. PCR amplification of the codon optimized L1-gene and pCM433        vector;    -   2. PCR amplification of the S-layer upstream and downstream        flanks. All reactions are done with a Q5 high-fidelity DNA        polymerase;    -   3. Gibson assembly and transformation into E.coli NEB 5-alpha        are performed using NEBuilder HiFi™ DNA assembly kit (NEBlabs);    -   4. Selected clones are validated by PCR (with Tag-polymerase,        Invitrogen) and sequenced (at Eton Bioscience Center);    -   5. Validated plasmids are subcloned into E.coli S17-1 via        transformation;    -   6. Biparental mating with M. alcaliphilum and clone selection is        set up as described by Ojala et al. [2011];    -   7. Genotype characteristics is validated by PCR;    -   8. Phenotype characteristics of new traits is evaluated via        scanning electron microscopy (SEM), lipase activity (rhodamine B        assay) and SDS-PAAG electrophoresis.

Example 2: Exemplary Methods and Compositions

This example provides exemplary methods for making compositions andbacterial cells as provided herein, and practicing methods as providedherein. Provided herein are new mitigation strategies for effectiveconversion of atmospheric greenhouse gases (e.g., CO₂ or methane) tonext generation chemicals which are a new technology for thereduction/stabilization of global warming. In alternative embodiments,methods provided herein comprise use of biological systems (microbialcells or enzymes) as catalysts for conversion of e.g., CO₂ or methane.

Methanotrophic Strain Best Suited for Biotechnological Exploration

Two methanotrophic cultures were established as the most promisingindustrial strains: Methylomicrobium alcaliphilum sp. 20Z andMethylomicrobium buryatenses 5G [Ojala et al., 2011; Kalyuzhnaya et al.,2015; Puri et al., 2015; Strong et al., 2016]. While M. buryatenses 5Grepresents a fast-growing methanotroph (Td=3h), M. alcaliphilum sp. 20Zwas found to be more stable at high-cell density. Furthermore, thelatter strain has a greater potential for accumulation of extractableproducts (ectoine, glutamate, sucrose). Based on those characteristics,M. alcaliphilum sp. 20Z was selected.

Genomes of both M. alcaliphilum sp. 20Z and M. buryatenses 5G weresequenced (see e.g., Vuilleumier et al., 2012). Genetic tools forefficient metabolic engineering of the strains were developed oroptimized (see e.g., Ojala et al., 2011; Puri 2015; Henard et al.,2016). The current toolbox includes: vectors for gene knockouts(incorporated via bi-parental mating or electroporation); vectors forheterologous expression with low, intermediate and high levels ofexpression; and vectors with tunable promoters. Provided is awhole-genome reconstruction of the M. alcaliphilum sp. 20Z metabolicnetwork, which is refined via metabolomics on cells grown in liquidculture, providing a computation framework for additional optimizationof metabolic pathways in producing traits.

Ectoine: Commercial Potential and Production in Methylomicrobiumalcaliphilum sp. 20Z.

Provided herein are methods for making ectoine((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), which isa well-known microbial compatible solute. Ectoine can be used as achemical chaperone for industrial enzymes or pharmaceuticals, acryoprotectant, a hydrator in skin-care products, a cell stabilizer formedical treatments, and a crop-protecting agent [Graf et al., 2008;Pastor et al., 2010].

Methylomicrobium alcaliphilum 20Z copes with high salinity in its growthmedium by accumulating ectoine (up to 8% CDW), glutamate, and sucrose(up to 12% CDW) as major osmoprotective compounds. This strain was usedas the most promising culture for ectoine production, see e.g., Totsenkoet al, 2005. The ectoine biosynthesis pathway in M. alcaliphilum 20Z issimilar to the pathway employed by halophilic/halotolerant heterotrophsand involves three specific enzymes: diaminobutyric acid (DABA)aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoinesynthase (EctC) (see e.g., Reshetnikov et al., 2011), as illustrated inFIG. 3 . The ectoine biosynthetic gene cluster is organized as oneoperon (ectABCask) and is controlled by a negative transcriptionalregulator (EctR1, marR-family). The EctR 1 protein represses theexpression of the ectABC-ask operon from the ectAp₁ promoter.

Baseline parameters and initial rates/titer of ectoine production forthe wild strain (or wild type, WT) include: biomass yield (Y: 0.46),growth rate (0.09 h−1), O₂/substrate ratios (1.54), ectoine titer (1.9%DCW).

Strains Lacking the ectR-Regulator and Expressing LipL1 Were Constructed

A set of strains lacking the ectR-regulator and expressing LipL1 wereconstructed. The strain lacking the ectR-regulator showed an ectoinetiter similar to the WT; however, the strain demonstrated overproductionof a compound X, which was identified as a product of ectoinedegradation. The deletion of the doeA-gene encoding ectoine hydrolase inthe ΔectR background (Strain 20Z^(R)ΔectRΔdoeA) eliminated compound Xaccumulation and led to an increased production of ectoine (2.4% DCW). ALipL1 expression plasmid (P_(SL)-L1 construct) was subsequentlyincorporated into 20Z^(R)P_(SL)-L1ΔectR (to create TWC #G2) and20Z^(R)P_(SL)-L1ΔectRΔdoeA (to create TWC #G2-3) which express LipL1.The specific activity of methanotrophic lipase is 1.2 U g⁻¹ CDW.

The growth rates of the strains TWC #G2, TWC #G2-2 and TWC #G2-3 aresimilar to WT. The strains TWC #G2-2 and TWC #G2-3 showed elevatedectoine (2.4% and 3.1% DCW, respectively), which corresponds to aproduction rates of 2.2 and 2.8 mg g⁻¹ CDW h⁻¹, respectively. Thechemostat culture of TWC #G2-3 displays similar properties to WT growthkinetics and shows ectoine production as 3.3 mg g⁻¹ CDW h⁻¹. Thus TWC#G2-3 shows 1.7/1.8-fold improvement. The specific activity ofmethanotrophic lipase in TWC #G2-3 is 1.2 U g⁻¹ CDW.

Expression and purification of LipL 1 protein. LipL was expressed andpurified. Twelve mg of the protein with the specific activity of 589U/mg were produced.

TABLE 1 List of exemplary genetic modifications in M. alcaliphilum sp.20Z^(R). Genetic alteration Strain Locus tag Phenotype IP/publicationDeletion: ΔectR::kan MALCv4_32 No growth defects. MustakhimovTranscriptional 51 Overexpression of the et al., 2010 regulator MarRectABC-ask operon. family Deletion: Sucrose- Δsps::kan MALCv4_06 Noaccumulation of sucrose WO2015058179 phosphate 14 is observed. No growthA1 synthase defects Deletion: Δglg1::kan MALCv4_35 Glycogen accumulationis WO2015058179 Glycogen synthase 07 reduced (10% of WT). No A1 1MALCv4_35 growth defects 08 Deletion: Δglg2::kan MALCv4_35 Glycogenaccumulation is WO2015058179 Glycogen synthase 02. reduced (5% of WT).No A1 cluster 2 MALCv4_35 growth defects 03 MALCv4_35 04 Deletion:Δams::kan MALCv4_06 Significant decrease in WO2015058179 Amylosucrose 17intracellular glycogen A1 accumulation and increase (15-20%) in sucroseaccumulation. No growth defects Deletion:EPS Δeps::kan MALCv4_06 TBD Newbiosynthesis 18- MALCv4_06 19 Deletion:ectoine ΔdoeA MALCv4_32 Thestrain was constructed in New hydrolase 46 20Z^(R) ΔectR backgroundOverexpression of Trait TWC#G1 No growth defects. Culture New lipase20Z^(R)::SL_(Cter)-LipL1 displays lipase activity (1.2 U g-1 DCW) EctRdeletion 20Z^(R) ΔectR Unmarked 20Z^(R) ΔectR strain New wasconstructed. No growth defects. Accumulation of products of ectoinedegradation. No/mild increase in ectoine accumulation. EctR deletion andTrait TWC#G2 No growth defects. New overexpression of 20Z^(R)ΔectR::SL_(Cter)-LipL1 Accumulation of products of lipase ectoinedegradation. No/mild increase in ectoine accumulation. EctR deletion andTrait TWC#G2-2 No growth defects. Increase New DoeA deletion 20Z^(R)ΔectR ΔdoeA production of ectoine (1.6 fold increase). EctR deletion andTrait TWC#G2-3 No growth defects. Increase New overexpression of20Z^(R)::SL_(Cter)-LipL1 production of ectoine (1.7 lipase ΔectR ΔdoeA(C-term) fold increase). Culture displays lipase activity (1.2 U g-1DCW). Overexpression of Trait TWC#G2-4 Improved growth (25% New, relatedIcl/ms 20Z^(R)::P_(hps)-icl-ms higher growth rate). to ectoine.Increased production of ectoine. Overexpression of Trait TWC#G2-v5 andv6 improved ectoine production New icl/ms 20Z^(R) ΔectRΔdoeA:P_(hps)-icl- (5.2%). No growth defects. ms Overexpression ofTraits TWC#G3-TWC#G5 No growth defects. 6.2% New ectoine20Z^(R)::Phps-ectABC DCW ectoine biosynthesis 20Z^(R)20Z^(R) SL_(Cter)-LipL1ΔectR ΔdoeA::P_(opt)- ectABC Multiple deletions TWC#G6-TWC#10Traits with improved ectoine New 20Z^(R) SL_(Cter)-LipL1 ΔectRproduction Δsps Δglg1 Δglg2 Δeps ΔdoeA::P_(opt)-ectABC-icl-ms Multipledeletions Traits TWC#20 Traits with improved ectoine New 20Z^(R)ΔectRΔsps Δglg1 production Δglg2 Δeps ΔdoeA::P_(opt)- ectABC-icl-msExpression of TWC#G11 20Z^(R)::SL_(Nter)- Lipase protein is expressedNew LipL from N-ter LipL1 and excreted Multiple deletions TWC#G12Terminated. New system for New & Overexpression 20Z^(R) SL_(Cter)-LipL1ΔectR LipL expression should be of lipase and Δsps Δglg1 Δglg2 Δepsconstructed. ectoine ΔdoeA::P_(opt)-ectABC:icl- ms::SL_(Nter)-LipL1Expression of TWC#G13 20Z^(R)::SL_(Nter)- No growth defects. LipL NewLipL from N-ter LipL1-Ssp DnaB mini-intein accumulates in cytosol withintein mostly. Expression of TWC#G14 20ZR::SL_(Nter)- No growth defects.New LipL from N-ter LipL1-Mxe GyrA Expression of GFP20ZR::GFP-12Cter_(SLP) Protein is expressed and New with SLP C-termexcreted into growth medium fusionInitial Cultivation Parameters for M. alcaliphilum 20Z^(R) in BatchCultures.

Strain and growth media: M. alcaliphilum 20Z^(R) cells were grown usingmodified P media (g/L): KNO₃, 1; MgSO₄×7H₂O, 0.2; CaCl₂×2H₂O, 0.02;NaCl, 30; trace solution, 1ml/L (Table 2); and supplemented with 20 ml/Lof phosphate solution (5.44 g KH₂PO₄; 5.68 g Na₂HPO₄) and 20 ml/L of 1Mcarbonate buffer.

TABLE 2 Trace solution composition. Trace solution (1000x) g Na₂EDTA 5The solution was autoclaved at FeSO₄ × 7H₂O 2 121° C. for 20 minutes andstored ZnSO₄ × 7H₂O* 0.3 at room temperature for up to MnCl₂ × 4H₂O 0.036 months. CoCl₂ × 6H₂O 0.2 CuSO₄ × 5H₂O 1.2 CuCl₂ × 2H₂O* 0.5 Na₂O₄W ×2H₂O 0.3 NiCl₂ × 6H₂O 0.05 Na₂MoO₄ × 2H₂O 0.05 H₃BO₃ 0.03 *The growthmedia has higher concentrations of CuCl₂ and ZhSO₄ compared to ourpublished media (modified from Demidenko et al., 2017).

Cultivation

Culturing was carried out in either closed vials (50 ml culture in 250ml vials, with shaking at 200 r.p.m.) or bioreactor cultures (fed-batchor turbidostat). Two types of bioreactors were used: 1) a DASBOX™(DASbox) mini bioreactor (0.5 L working volume; 200 ml culture) with twoindividual bioreactor units, each having automatic temperature, pH, andDO controls, a sample port for measuring OD, and a coupling to aBLUESENS™ (BlueSens) sensor system for simultaneous measuring off-gases(CH_(4,) O₂, and CO₂); or 2) a 2.7 L bench top BIOFLO (BioFlo) 110™modular bioreactor (New Brunswick Scientific, Edison, NJ, USA). Cultureswere also grown as batch cultures (in triplicate). In all cases wemeasured CH_(4,) O₂, and CO₂ in the headspace to determine consumptionand production rates and the O₂/substrate utilization ratios using anSRI GC system. In addition, samples were taken for measuring ectoineconcentrations in cell biomass by HPLC. The data were analyzed to assessyield (Y), growth rate, and O₂/substrate ratios (Table 3).

Dry cell Weight (DCW) Measurement

Cultures (150 ml) from bioreactors were centrifuged to collect thebiomass. After careful removal of the liquid phase, tubes of knownweight with biomass were weighed (to obtain wet cell biomass weight),lyophilized overnight using a LABCONCO™ freeze-dry system and weighedagain. The observed DCW parameters were as follows: 1 L of cell culturewith OD=1 corresponds to 0.336±0.025 g CDW.

FIG. 4A graphically illustrates data showing the growth of M.alcaliphilum 20Z^(R) in a DASBOX™ (DASbox) mini bioreactor (0.5 Lworking volume; 200 ml culture), as batch (0-20 hours (h)) and chemostatmode (20 h-90 h). Steady-state was reached at 60 -80 h. B—C. O₂ and CH₄consumptions and CO₂ production in steady-state for bioreactor replicate1 (FIG. 4B) and 2 (FIG. 4C).

HPLC Protocol

Twenty mg of lyophilized biomass was re-suspended in 200 μl of water.One ml of 0.2M sodium citrate buffer (pH 2.2) was added and quickly (15sec) sonicated to re-suspend. The mixture was allowed to sit on thebench at room temperature overnight (18 h) and then sonicated again (15sec). After centrifugation for 10 min, the clarified lysate was filteredthrough 3 kDa centrifugal filter units (Millipore) and the filtrate wasanalyzed by high performance liquid chromatography (HPLC). Ectoineconcentrations were determined by using a previously published assay (Heat al., 2015), i.e., an isocratic mobile phase of acetonitrile and water(70:30 v/v), flow rate of 0.5 ml/min and a detection wavelength of 210nm. Samples (10 μl) were chromatographed using an Agilent 1100™ HPLCsystem equipped with a NUCLEOSIL NH2-HPLC™ column, 5 μm particle size,25 cm×4.6 mm (Macherey-Nagel). Pure ectoine purchased from Sigma wasused as a reference (FIG. 3A).

FIG. 5A illustrates a chromatogram of 1 mM ectoine solution; ectoinepeak area=8497; FIG. 5B illustrates a chromatogram of 20ZR cell extract;ectoine peak area=18066 (corresponding to 370 ug of ectoine per 20 mg ofdry cells, or 1.85%).

Results

A bench-scale New Brunswick BIOFLOW (Bioflow) 310™ bioreactor was usedto accumulate cell biomass. A DASBOX™ (DASbox) mini bioreactor systemwas used to generate performance parameters for continuous culturesgrown on methane. The parameters measured from these growth conditionsinclude cell dry weight, CH₄ and O₂ uptake rates, glycogen content, andexcreted organic acids.

The parameters for continuous culture conditions and catalystperformances are shown in Table 3, below. A maximal growth rate of 0.13hr⁻¹ was obtained under fed-batch conditions using our standard gasmixture. The specific growth rate in the continuous culture was 0.09-0.1hr⁻¹ (see FIG. 4 ) at DCW 1.15 g/L.

TABLE 3 Baseline parameters for M. alcaliphilum 20Z^(R) grown in abioreactor with methane as a source of carbon/energy. Parameter SD Gasinput 5% CH₄:3.5% O₂:N₂ balance Gas flow 1.6-1.7 L/h Bioreactor volume0.2-2 L Growth rate (h⁻¹) 0.09 0.01 Methane consumption (mmol g 12.2 0.9DCW⁻¹ h⁻¹) Oxygen consumption 19.7 3.3 (mmol g DCW⁻¹ h⁻¹) CO₂ produced(mmol g DCW⁻¹ h⁻¹) 4.6 0.9 Y CO2 (%) 0.37 0.05 Y (biomass) 0.46 0.02O₂/CH₄ 1.54 0.04 Ectoine (% DCW) 1.86 0.07 Productivity (g ectoine g⁻¹DCW h⁻¹) 0.0016 6.3 × 10⁻⁵Construction of a Methanotrophic Strains Lacking ectR-Regulator andExpressing LipL1.

Construction of 20Z^(R)ΔectR strain lacking ectR-regulator. The strainwas constructed and tested for ectoine production:

Strain construction. Plasmid pCM433kanT carrying approximately 800 basepairs (bp) of sequences flanking ectR gene was constructed andintroduced to 20ZR strain by biparental conjugation. After mating,single-crossover, kanamycin-resistant clones were plated on rifampicinto counter-select against E. coli. Then, to select for Kan-sensitivedouble crossover clones with a deleted ectR gene, single-crossoverclones were passaged on plates with 2.5% sucrose and the resultingcolonies were PCR-genotyped for the absence of ectR followed bysequencing (underlined sequences, as described below).

Results. Amounts of ectoine in 20Z^(R)ΔectR strain are similar to theparental (WT) strain, see FIG. 6 (illustrates HPLC analysis of 20Z^(R)wild-type (batch culture)) and FIG. 7 (illustrates HPLC analysis of20Z^(R)ΔectR strain (batch culture). However, the peak adjacent toectoine (at 17.8min) was significantly enriched in the ΔectR strain(FIG. 7 ). It was suggested that the peak might be an intermediate ofthe ectoine degradation, thus an additional mutation, knockout of theectoine hydrolase (doeA) gene, was generated.

Construction of the 20Z^(R)ΔectRΔdoeA Strain.

Strain construction. The strain 20Z^(R)ΔectR was used as the parentalstrain. The ΔdoeA knockout was constructed the same way as for the ectRdeletion. The selected clones were PCR-genotyped for the absence of thedoeA gene followed by sequencing.

Results. HPLC analyses of the cell extracts showed increased level ofectoine in the 20Z^(R)ΔectRΔdoeA strain (26% more than in WT, Table 4)and no ectoine degradation intermediate (compound X) was observed, seeFIG. 8 (illustrates HPLC analysis of 20Z^(R)ΔectRΔdoeA strain (batchculture)).

Construction of the TWC #G2 and TWC #G2-2 Strains Expression LipL1.

Strains for simultaneous production of lipase (as a fusion with S layerprotein) and ectoine 20Z^(R)P_(SL)-L1ΔectR (TWC #G2) and20Z^(R)P_(SL)-L1ΔectRΔdoeA (TWC #G2-2) have been made.

Strain construction. EctR and doeA genes were introduced into WT and TWC#G1.

Results. HPLC analysis reveals the highest levels of ectoine, 160% morethan in WT 20Z^(R), Table 4, FIG. 9A-B (illustrates HPLC analysis ofHPLC analysis of 20Z::P_(SL)-L1ΔectR (FIG. 9A) and 20Z^(R)P_(SL)-L1ΔectRΔdoeA (FIG. 9B) strains (batch cultures).

Additional Genetic Strategies for Improving Ectoine Production.

As an additional way to improve ectoine production, an isocitratelyase/malate synthase fusion was expressed in the 20Z^(R) strain underhps promoter (P_(hps)). The expression of the construct was expected toprovide an additional route for oxaloacetate production, a keyintermediate in ectoine biosynthesis. As expected, the level of ectoinein the strain 20Z^(R)::P_(hps)-icl-ms was increased (26% more, Table 4)compared to the wild type strain. Incorporation of thepCM132::P_(hps)-icl-ms producing plasmid into strain TCW#G2-2 strain isin progress.

Batch Culture Cultivation.

Growth characterization of the strain was done as described for WT. Allbatch cultures showed the same growth rate as WT cultures. Ectoineconcentrations were estimated as described in Table 4. Each additionalexperiment included WT cells as a control.

TABLE 4 Ectoine titer and production rate in genetically modifiedmethanotrophic traits: Averaged data (n = 2-6) for productionProductivity Strain Strain Ectoine, % of (mg g⁻¹ genotype name DCW % ofinitial CDW h⁻¹) WT- ref^(R) 20Z^(R) 1.9 ± 0.04 100 1.6 ΔectR 20Z^(R)ΔectR 2.0 ± 0.15 100 1.6 20Z^(R)P_(SL)- TWC#G2 2.6 ± 0.06 100 2.3L1ΔectR 20Z^(R)ΔectRΔdoeA TWC#G2-2 2.4 ± 0.3  125 2.2 20Z^(R)P_(SL)-TWC#G2-3 3.1 ± 0.06 160 2.8 L1ΔectRΔdoeA 20Z^(R)::P_(hps)-ms- TWC#G2-42.4 ± 0.02 125 2.7* icl *The strain has improved growth rate (wasrecalculated to specific growth rate in the continuous culture as 0.12vs 0.09 h⁻¹)

Cultivation in Mini-Bioreactor in Continuous and High Cell Density BatchModes.

Cultivation of TWC #G2-3 was performed in a DASBOX™ (DASbox) minibioreactor (0.5 L working volume; 200 ml culture with two individualbioreactor units. Gas input and operational parameters were the same wayas described for WT strain. Collected data are summarize in Table 5 andshown in FIG. 10A-C (FIG. 10A illustrates growth of M. alcaliphilum20Z^(R)P_(SL)-L1ΔectRΔdoeA (TWC #G2-3) in DASbox mini bioreactor (0.5 Lworking volume; 200 ml culture), as batch (0-65 h) and chemostat mode(65 h-120 h); Steady-state was reached at 90-100 h. FIG. 10B-Cillustrates O₂ and CH₄ consumptions and CO₂ production in steady-statefor bioreactor replicate 1 (FIG. 10B) and 2 (FIG. 10C)).

The strain TWC #G2-3 grown in continuous culture in mini-bioreactorproduce twice the amount of ectoine as WT (Table 5). The ectoineproductivity was calculated as was 3.3+0.3 mg h⁻¹g⁻¹ CDW.

TABLE 5 Parameters for TWC#G2-3 grown in a bioreactor with methane as asource of carbon/energy. Parameter SD Gas input 5% CH₄:3.5% O₂: N₂balance Gas flow 1 L/h Bioreactor volume 0.2 Growth rate (h⁻¹) 0.09 0.01Methane consumption (mmol g DCW⁻¹ h⁻¹) 8.6 1.2 Oxygen consumption 11.61.6 (mmol g DCW⁻¹ h⁻¹) CO₂ produced (mmol g DCW⁻¹ h⁻¹) 3.1 0.4 Y CO₂ (%)0.37 0.45 Y (biomass) 0.59 0.13 O₂/CH₄ 1.35 0.01 Ectoine (% DCW) 3.10.07 Productivity (g ectoine g⁻¹ DCW h⁻¹) 0.0033 2.9 × 10⁻⁵

Expression and Purification of LipL1 Protein.

Purification of lipase after expression in E.coli BL21 (DE3).

Strain Construction.

Codon-optimized sequence of LipL1with N-terminal His₆ tag was clonedinto pET21 plasmid under T7 promoter; the construct was introduced intoE.coli BL21(DE3) strain.

Expression and Purification.

Cells were grown in 300 ml of LB with ampicillin (100 μg/ml), at whichpoint lipase production was induced by addition of IPTG (0.5 mM final)at OD600=0.5 and continued for 7 h at 37° C. Cells were collected bycentrifugation. For purification, cells were lysed by French Press(purifications 1 and 2) or by sonication in the presence of 0.5% TritonX-100 (purification 3), clarified lysate was loaded to Talon resin(Clontech) for one-step purification by metal affinity chromatography.After washing of the resin and elution of lipase with 200 mM imidazole,lipase prep was dialyzed against 20 mM tris-HCl (pH 8.0) and 100 mM NaClbuffer followed by addition of glycerol to 50% w/w and stored at −20° C.

Activity Assay.

Activity of the isolated lipase has been confirmed on Rhodamine B platesand by p-nitrophenoldecanoate assay. One unit was defined as the amountof enzyme that released 1 μmol 4-nitrophenol.

Purity Validation.

Purity of the purified lipase was checked by electrophoresis on SDS-PAAG(12% mini-Protean TGX™ gels, Bio-Rad) according to manufacturer'sprotocol. Gels were analyzed and quantified with IMAGELAB (ImageLab)4.1™ software (Bio-Rad) and are shown below (FIG. 7 ); in all 3 casesonly the protein band corresponding to LipL 1 is visible, no other(contaminating) proteins are present.

Three sets of LipL1 expression and purification were performed. Intotal, about 12 mg of pure L1 lipase were isolated.

TABLE 6 Summary of LipL1 protein preparation Volume Protein Specificactivity* Preparation ml mg U/mg P# 1 1.5 3 630 P# 2 2 7 400 P# 3 2 21200 Total 5.5 12 589 *1 unit (U) is the amount of enzyme that catalysesthe reaction of 1 umol of substrate per minute.

Lipase Production in Methanotrophic Strain TWC #G1, TWC #G2 and TWC#2-G2:

Different constructs have been made for production of lipase fromplasmids (under different promoters) and from genomic DNA as a fusionwith S-layer protein. All of the strains have been shown (qualitatively)to produce active lipase by both Rhodamine B and p-nitrophenoldecanoateassays. The specific activity of methanotrophic lipase in TWC #G2-3 is1.2 U g⁻¹ CDW. FIG. 11 illustrates LipL1 preparations.

Construction of Strains TWC #1, TWC #2 and TWC #2-2:

The coding sequence for LipL lipase was introduced into the 20Z genomeas C-terminal fusion with S-layer protein of 20Z^(R). A HRV3C proteaserecognition site was placed in frame between the S-layer protein andlipase sequences to allow protease cleavage of the fusion polypeptideand release of the free lipase. The codon-optimized LipL lipase sequence(synthesized at GENSCRIPT™ (GenScript)) with the HRV site was introducedby PCR. Plasmid pCM433kanT carrying approximately 800 base pairs (bp) ofsequences flanking the fusion site of S-layer protein was constructedand introduced to the 20Z^(R) strain by biparental conjugation. Aftermating, single-crossover kanamycin-resistant clones were plated onrifampicin to counter-select against E. coli. Then, to select forKan-sensitive double crossover clones with inserted lipase gene,single-crossover clones were passaged on plates with 2.5% sucrose andthe resulting colonies were PCR-genotyped for the presence of lipasefollowed by sequencing.

Genomic region of ectR (MALCv4_3251) with upstream and downstream flanks.The genetic region deleted in ΔectR strain (ectR gene) is underlined)(SEQ ID NO: 1):gaaccgctttgaacggcccaaccttcaccagcatttcggtttcatgttgatctgcaaaatggcgtgtcttatcaaacatcacagcactgccaatttgagtgtccagtttttttgccagcccctcaaacagagcccatgaggccttattattcggagtaatggtcgtctcgattcggttaatatcctgattgaccggccgcgccagtatggctttcagcatccgcgtggcaagcccttggccacgggctttttcgccgacagccacctgccagacaaacagcgtatccggacgttgcggaatacgataacccgagacaaaaccaaccaactcatcgccaattttggccgccaccgccgtttcagaaaaatggctgctctgcagcaaattgcagtacatcgaattgggatccaggggcgggcatttgctaatcagccgatgcacctgcgctccgacttcggcagtaggctggctaagtgtaataatcggcaaggcagttttatcaggcaacatataaataactctattatttagatttctgtgcaattaactcggctttaactgaataagccgggctcgaatttgattttttatggccatcagcacgaatattctggcttcattgaaaaacataatatatagtacactaaataatttaaatgtccaggccgcgtacttttgccttagattaattagatgtcatatcaaattatgcctttcgaacttcaaatttcggtagcgccctaggatgcgccgggcggagcaccgaattttgttcttcgagggtatacaataatggctttgtaacgcgacggcctctatttcaatgattggtgatcaatgatgcaaaacccacaaccgcacgcccctcattcgctggatacgctcgacttgaatccggttgaaaaggaacatttgctgaatcaaattgaagaagtactggtcgcgttacgtagagtgattcgcgccaccgatttacactcaaaatatctggcaaaaaccactagcctgaccgcaccgcagattcttttgttgcagacactgcgcgccaaaggtcaactgaccattggtgagctagctcaggacatgagtctcagccaagcgactgtgacaacaattctggatcgcctggaaaaacgtcaattggtgttccggcagcgctcccagactgataaacgaaaagtccatgtctatatgacggaggcggccacggaaatgctaataaacgcccctatccctttgcaggatcgctttacgcgagaattcagtaaactacaggaatgggaacaattgatgattattgcatcactgcaacgtgtcgctcagatgatggacgcgcagaacatccctgtcgctaaagaagcgtttgattttccggtttaagctctaataattcagctcagctgcaacccgcatcacgctttttcccaagctccagcttgggaaaaacaccccggaagctccagcttccagaaaccgagataacctccgcacattctcaatcaaccccgactcgctcgttcaatctttttcctgattagcaagatgcttcaacttgctgaagccaattgtccgagcagtggccagtcgtagccacttctgcgggacgggttatttaacccatccccaacgtttcggtttgccctaaacatttcggctgacttcggccaaagtcaaaacgtttaggacggggctgcaaaccccgtcctgctaaggatatgctggttttcgggctttagctgaagaaacttgctaatcaggatcttttttgattgtcgggaagctagagcttcctgaatagattacccaagccggatcgctcgccgctatacacaaatatcggtaaacttgctaaacagaggtcgccgtatacttggaagcggtgctgtttgataaatctgcggatattggacatcagtggcttcgaagtcgggaacgttgggcgataaaaaatggtatcgaggcctcattggttaagattgcaaaagggtaatttttgacttatgtataacgatgaggtaactattcagtcccccggcaattatcgttcccacgctctgcgtgggaatgcctgagtaccgctccagcggtacgagacgctagagcgtctcggtcttcattcccacgccggagcgtgagaacgataggcggtgtgaataattacacgatgagagctggagcttgggtaacagcacaaaggcatatttagcctagttgcGenomic region of doeA (MALCv4_3246) with upstream and downstream flanks.The genetic region deleted in ΔdoeA strain (doeA gene) is underlined(SEQ ID NO: 2):gtagcaagccttgcgtagagattattgccgggtgtaaaggcgtctatgctttcgaggtgttcgatcaaaacatggccggcgagattcacaattacgatcgggaaatcctggggcaaattcgccgcttcagagcgcatattgtttccaaagatattaatgccaataccattactcattatttgtctgccagtctgaaaacaattcgccgcatccgcgatgccttgcaagaaatctatccggatgccgaaatcaatcagcaaaaagtttcgattgtttccgccatcggcagtgacatgaaaattcccggtattctggccaaaactgtctcggcgctggcagagaagcagatcagcgtgcttgcaatgcaccagtcaatgcgtcaagtcgatatgcagtttgtgattgatgaagatgcctacactgatgcgatgaaaagtttgcattgtcatctggtggaggtccatgatcacggcattgcaatatgcctcgcgtcctgattgtactgatgtttcttacccctaaatacggggaaatttctcaaactgggaattgctgccaaagaaatgaaaatgccttgcgtcttcagtcttgcgctgaacgacaaggaagcgcataaggcttaaagcgtttaccactcgaccgctgaagcggtagcggattaaggcgagtcgcaagtcaattttcgtccaaggttaggttgttcatggcaagtcagtcgagcaatgaatgacttaaccgtctatttttcaataaactgaagatgtacgggtaagccctgcgtaagttgggaatggccgatgatcgagggctatctgttgtcgcgaagtctttaatcaaaaaaatgggtttaatattcaatgattaccgagaatgccgcacagtccgaacaaagtgaagatttttatcaatcacgtaacggtagtaagccgaaaataattccgcgcgtagacccggtagtttatgcgcaaacagctaatccaggtctcattgcagaggacttgcaagcacgttatgagcaacaaggttttcttgttattgataatgtttttaatgagagggaggtcgactgtttcaagcaagagctcaaacgcttgaacgacgatgaaaagataaaagcctcggcggaagcgataactgaattatccagcgacgaactccgttcactatttaaaattcatgaagtcagtccggtttttaaaaggttagctgccgataatcgattagcgggactggctcaacatcttttgaacgaccgggtttatattcatcagtcgcgcttaaactataagccgggttttcgcggcaaggaattttactggcattcggactttgaaacttggcatgtagaagacggtatgcctagaatgcgtgcgctcagcatgtccattattcttaccgaaaacgatcagcataacgggcctttgatgttggttcccggatcgcataaaaaatttgtcgtttgcgaagaggaaacgccggaaaatcattattcggtctcgttgaaaaagcaggagtacggcatacccagcgatgaatgcttggctagcttggttgccgatggcggcatcgtatcggccaatggaaaacccggcagtgtcttgattttcgacagtaatgtcatgcacggttcgaatagtaatatcactccatggcctcgctcgaatctctttttcgtctataacgcgatcaataatcgagtaacatggccgttttgcggtttattgccgcgtcctgaatatctttgcagtcgcaagaatatacgagttatcgaaccgcggccttttatcgcggccgccgatcaattgatatatgcttagaatgttaataatgttgatcgtgctggcgccctgttccgtgttgggcgagagcgtcaacgatgaagcagaggttcaagagcgcttagatgcggttgaatctttggataagcctttatatagtccgttcatcgagcgctatatgctggatgaactcaaacaattgcgtatggacatggcagcgcagaggaatgagctgattcagcaaattgtggatagagagcttagctcggtcgatagaggcgttacttacgccactaatactgtcacatattttttctacttgattgccggtgccagtaccattttggtgttgctgggttggacctcgctcagagatatcaaagagcgtgtgcagtccatggcggataagaaagtatcgaaactggtccatgaatacgaagagcgcttggcaattgtcgaacaacaactcaacaaggaagcacaattgattgagaaaatcggcgaggatatcgggcggacgcaagatgtgcaatctctctggcttagagcaggtcaagcaggcagcttggccaataaaatcgccatctacgatcaaattttaaaattgcgtcccgaggattgcgaagcattgacttataaggccgatgcggtactcgatatgggcgagccgcagtgggccgtcaatttatgtcagcaagcgttgaaaatcgaccctgaaaacggccatgctttttaccaattggcttgtgcgtataccgcattggatcaatatgaagaggccgttaactgtttatccgaagccttggcgcgtaccgaggattatcgcgataagtttgccgatgaccccgcgctgcaagcgttaaaaggttttgagccgtLipL sequence (His6 tag is underlined) (SEQ ID NO: 3):ATGGGTCATCATCATCATCATCATCTGGAAGTCCTGTTTCAAGGCCCGATGGCCTCGCCGCGTGCGAACGATGCGCCGATTGTGCTGTTACATGGTTTTACGGGCTGGGGCCGGGAAGAAATGCTGGGTTTCAAATACTGGGGCGGCGTCCGCGGCGATATCGAACAATGGTTGAATGATAATGGCTATCGCACCTATACCTTGGCCGTCGGCCCGTTGTCGAGCAATTGGGATCGCGCGTGCGAAGCGTATGCCCAATTGGTCGGCGGCACCGTCGATTATGGTGCCGCGCATGCCGCGAATGATGGCCATGCCCGCTTTGGCCGCACCTATCCGGGCTTGTTGCCGGAATTGAAACGCGGCGGCCGTGTCCATATCATTGCCCATAGCCAAGGCGGCCAAACGGCCCGTATGTTGGTCTCGTTGTTGGAAAATGGCAGCCAAGAAGAACGCGAATATGCCAAAGAACATAATGTCTCGTTGAGCCCGTTGTTTGAAGGCGGCCATCGCTTCGTCTTGTCGGTCACCACCATCGCCACCCCGCATGATGGCACCACCTTGGTCAATATGGTCGATTTTACCGATCGCTTTTTCGATTTGCAAAAAGCCGTCTTGGAAGCCGCCGCAGTCGCGTCGAATGCCCCGTACACCAGCGAAATTTATGATTTCAAATTGGATCAATGGGGCTTGCGTCGCGAACCGGGCGAATCGTTTGATCATTATTTCGAACGCTTGAAACGCTCGCCGGTCTGGACCAGCACGGATACGGCCCGCTATGATTTGAGCGTCCCGGGCGCCGAAACCTTGAATCGCTGGGTCAAAGCGTCGCCGAATACCTATTATTTGTCGTTCAGCACCGAACGCACCTATCGTGGCGCCTTGACCGGCAATTATTATCCGGAATTGGGCATGAATGCGTTTTCGGCCATCGTCTGCGCGCCGTTCTTGGGCAGCTATCGCAATGCGGCCTTGGGCATTGATTCGCATTGGTTGGGCAATGATGGCATCGTCAATACCATTTCGATGAATGGCCCGAAACGCGGCAGCAATGATCGCATCGTCCCGTATGATGGCACCTTGAAGAAAGGCGTCTGGAATGATATGGGCACCTATAAAGTCGATCATTTGGAAGTCATTGGCGTCGATCCGAATCCGTCGTTCAACATTCGTGCGTTTTATCTGCGTTTAGCGGAACAACTGGCGTCCCTGCGTCCGTGASequence of the S-layer protein (MALCv4 0971)-Lipase fusion incorporatedinto Methylomicrobium alcaliphilum 20Z^(R) chromosome. HRV protease siteis single underlined (i.e., is ctggaagtcctgtttcaaggcccg), and the lipasesequence is shown bold (SEQ ID NO: 4):atggcaacactctcagtggatatcgctcaatcctatatggagaccttacggtcctatgggcttgagtttaacataagacaaaccaacaacctgaccaatcggataattaaccgcttggaaaatcgcggccacacacctgagcaagtagcagactggcttatgagcagggttgcggttaaacagcaattgagaaaactggttaaacaaggcgaattggccgagtttgatctggatggcaatggccgcctcaatcgatctgaattgctaaatgcaatgtctgctctgtccgagacagcggtcgaagaagcgccggtcgaagatcccacgactcccaagcctccggccgatcctagcatcacgaccttaacgcttactgagattccgactcagcgtacggctacgttaaaatggaacaatgtcgatgccgatttggccatcgatttcatgcaagacgtactgaaactagatctaaatcgactaggctggatggaagacggtcaattgaccgtcaacatcgacaatatcgcgatcagcgattcggacagtaattctgatatcaacatcggtatggtcgatggcgaagaatttttgttcagcgtaaatacgccagtggcgctgtataccaatattatatttgatttgaagcaaaacgatgatgtcattcaaaccggcatcgtgctaacgccgaccgaaaacaacggcggttcgtttgaaaacggcattacctccgatgccgacaaccacatcatcgccggtcgtcctgaattgctgcacggcgcctacatcgacggcggcgggggctacaacacgttggaagtcgacatgaaaggcttctttgcgcagccgttccaactgttgaacatccaagagatccacgtacaaaacctcccgaatgtctacagtttcgatcaaacaattttcagcgacaccgaaggcgactattttgctaattttccaattcctacgaatttggatggcgatgatagcattcttgacttgagccgggccactagcctagaaagactggtcattaacgaagcacgctttcccggtagcgcaaatgccttaggcgacctctacctggtcggtatcaaagccgatgcggtcgcccgtctagaaggcaacttcaccgaagacgtaaacttgttctatggtcgcggcttgggtaatgcgatcaacctggaatttgccaatgtcacgatgagtgatagtgagggggggggtgaattggtgctgggtcataatgccggtaccgtgaatctgctttccgaaggtcgtctgaacgtcttggaatctgttgatttcggtagtttcctgcgcgaactcaccatcaccggtaccggagagttggttattgatgacgccctcgcattcgcctttggcgaagtacatatcgatgcctctgctaataccggtggtattcgcctcaaggtcgacagcgttgcagacggtagcagcctatccaatgaaatcggcttttctgcggttcttgacgaagtcaccatcaaaggctcacaaggtcgtgacgttatcgagatctccggcactgctgcaggcgtattgcttgacatcgacaccggtgctggccgcgacaccgttttcttgaccgacgatactttgagtgctggcgctggctcagtgatcaccggtgataatttgacagttgtggtgacagccaccgccgatctgcgtaaggccgatgttgttggcgttgaccgctttgtactgaatgcaggtcccgcagccgctggcaacctggttttgactcagacccaagtagaagccatggatgccggtgtgttcaccgcagcccataatactatcgcagttctgtcagtcgaaatcaccgaagcgggtacggttctgtcagatctgatcgatctttcggcactgagcagtgatgttaagctggccttcaatgttgttaaaggtgcaagccttgaaatgaccgctgaagaactgcataagtatgtagcttttgaaggcatcgatgcaactatggctggtcacctggtgatcaccggtgcgggtctgggctttgatcctgaagatcaatctgactacgatactggtggtacgattgccaattacggtcttacaccagaccaaaatatctccattattcgtgatccaaacggttttgagcgcccggcgcccgataccaacactgacatcctgaccattgataccacaggtggaatcaccatcggtgccaatgcactgtcagatgacgatgccttctcaaccaatgcaaccaccttgatcatcgaaggtgcaggtgatattacctttaatgcaccgttggaaatgttattagataactacaccatcgatttttccggcctgaccggcaatctcaatggcctgaccattctcgatttccagaacatcacggatggaaacgatccgagcgactggggccagattatcggtaaccccgatgttaatacccgtatcaacgtggtcattgaggacggtaaggaagttggtgatgacagtttaggcaatgccaatggcggcctcaagtcttccggtgtcgaaacctacgtagttctgggaactgtcggcgaaacctacaccttcaatgtttgtgataccacgcaaggccttgaagtcctcggtttccgtggtctgggtgatgtcacgttcaaccagatcaactggggcaccaacctgctgctggaaggcgatggctttgaaaactttggtgatattccgaaggcttttgccaacccgaaccaatccaacatcggcagcatcgaagccaactacttcttcgatggtgctactgtagatgttgccatcaacaaccagggtcaagctctgggcaccacctctacaggggcagcgcgtcccttagtggtagaaagcatcgtggtaaacggtgctgagaccgtaaacctggcaatcgaagacggcagcgcctggatcaaatctgttgatggctctgttctcgaagatctgaccgtcaccagtgatttccacgttacactctcgctgatcgctgccaacagcgacctcgaatctatcgatggatctggtgtcgtaggtgtcatggctctggagattagtgatgttgatggcgccggggatcctactgccctgaccgtagacctctcttctacagaactgagcggcatcgaccagatcgatctgggcccattggctgatctcactctgaacattgatcagattgaggacatcggcacagcaaacatcgcctttaccggtacggctgcacaagctcaaaatgatccagcgacgctgaacattggtcagtttgctgaacaagagtttgatattacgacagtcggtctggaagccggtgttgaactgggtactgttacctttgttgcgaatgcaggtgaaatcactatgcatccagacaccaacctgtccggtgcaactgcaatcgtgattccagaaggcacaaccgtgaatatgacggctgcgcagtatgagcagatcgtagatggtggaaacggtagcagcttctcaggcctcggtgtgttgaacatcaccgacctgctgggtgagcctgaactggatacaaatggtgatccgatcccaggagcctttgcttccgacatcaatctgtccggtgtacctgtcgaaatgatgggcagcatcagcctggcagcgggtgtcgatagcatgcgcctgacgggtaatatcggtatcgctcagtttgaagaaagggaaattgctgatcctgataatacagacttcgtatcggttcagacttcgcataattttcatgaactgaccggcgaaacgcagggcttcagctttgtgctggccgaagatcagaacctgatcttcaccacagaagcgcaggcccataaccgtgtggttgaaggcgatggctctaaagtcacgctggcgtttgctgtgctgctggactctcaggtcaacttcaatacgctgggcggcactcttgctgttgatggtctgaacctggcctactacagcgatttgaccgatcttgaagtgcttcaggccttggtagctggtactaacgttgagcagatcctgggtaatctggatgaaaatacagttgttcagatttctgagtttattgctggcgctaactttgctaatccgactttccgcgatgtggaagtgttggaaggtgtaactgttgccggcggtctggtcttcgagaacctgaacgatgagttaccggatagcctcaagctgactgaactgagtctcagcctgttgggtgatgcgactatcacaggtacggttgatattagtggtgcaccactgctggatcagggcttcgaaaccctgaccatcaactcgctgggtgatgatcccaacaccatcaataatgtcgtagcgacaggcaacgatctgatcgatgtcgtgatcaatgctgaacaggatctgtcagttcaaaccatcaccttgagctttgttccaaaaacacctggtcaaacatcagacgcaaccttgacagtcaatggtgatgcagatgtaaccatcaagacactggatagcactgatcctgatatcaacgtagttaacattgataacaacctgactggcggtgccacactcaccttcacaggtggttcagctgcattcgagggtgatgatacagatagcctgatccttaccggtgcaggtaacactgtatttgatactgaaggtgcaagtacgggtggtatcgactccgatagcctgtcactgatcgatgcttctgagcacaccggtgatctggacctgggtcgtatcatcagtgttgacgaagctaacttcagcctgctgacctccgccggtaatgcctcagctacgctgcaagccactatgaatgatcaaggctttaatgccgcggtcctggcttacaacgccgctctggcagcggatcctgttgttccgggaaacgttactgcagcgctaactgctctaactactgctgcaggtcttcttggctttgtagatgcaaatgaagatccgctggccttcactcaggctaacgcggctgatctgatcgcagagttccgtcctgaatggaactttgagctgggtgctaacaccgagctaaccatcgatggagatgacatcgttggagctgactttgttgccggtgccctggtgatatccggtggcaagttgatcatcgaaggtgaagtcgatctgcgtgatctggctgtactggacatctctgatgtcgagattgagctggccgcaggtgcacgtatcctgatgactgacgagcagttcgacgcgctggacaacgtcaccttctctggcccaggtcagacgctggaagttgatgatgcgctgctggctgagctttctatcgtcaacgatatcactgatattcgcggtgtcactgagattcagttggaagaaggcctggttgaagacatcaccatgacagctgagcaggcgcgtatcgcgactgtagtcgatgccgacggtaaccctgtcctggttgacttcgatgttgatccaactgtgatcgatgctgcgggtgaccctgttgaagccggagacttccgtacccttacaggttctgttgttaccgtcgaagtaacaggtaacgacgatctgaccgatctggctggcctgaatcgcattgaaatcgtcagtgatgatctggttgatctgaaagtggcactggatcttgatggtgctaacaaccaggttgccgcgttgcaaactgcagcgggtggagcgtttgacggcacattcttcgatgtgttgagcaacttcactgttgaagcaagctttgaggtgctgagccagtttgaccctgaaaccacgttgttcgttgccaatccgatcgtggaggatgtcaacttcgacatcgtacgtgatgtgaatggtgatgtgacttcagtcagcgtcagcggtggctcttcccttggtttcgcccagagcgatgccggcttccaggagttgttggaagccggtcaggtaactgaggtggtgttcgagaatgttggttcactcaacagcatccttgttagtggcaacttcgtcggtagctacgatgccggtggtattttctacgagagcacctttgagttcggcgcaaatgctggttcggttgctgaaggggtgggtacagatggcaacatcttcaccattgctgaattcacagccggtgcagcagccagtgatatccttgatttcactgccatgcctgttgataacacgaacactgctccagccactgggcatgagttcatcgcggtaggcactgaagctagcattggtgacgatgccaccatcattgtcttcacggcgggtgttgcggccgacgcagcaaccatcgtgacacagtttgctgatggtgcgggagatttccgttcagcagatgctactgcacgtaacgctgactttgctattgatagccagttgatcttcctgattgacgatggcgctggtaataccggtgtctggtattgggatgatacagttggtgctgttggcgatggtattgtcgatgctgatgagctttcgcagattgcccagttgactggagtcgtcactgccgagctgacggttgataacttcgtcctcgctctggaagtcctg tttcaaggcccgatggcctcgccgcgtgcgaacgatgcgccgattgtgctgttacatggttttacgggctggggccgggaagaaatgctgggtttcaaatactggggcggcgtccgcggcgatatcgaacaatggttgaatgataatggctatcgcacctataccttggccgtcggcccgttgtcgagcaattgggatcgcgcgtgcgaagcgtatgcccaattggtcggcggcaccgtcgattatggtgccgcgcatgccgcgaatgatggccatgcccgctttggccgcacctatccgggcttgttgccggaattgaaacgcggcggccgtgtccatatcattgcccatagccaaggcggccaaacggcccgtatgttggtctcgttgttggaaaatggcagccaagaagaacgcgaatatgccaaagaacataatgtctcgttgagcccgttgtttgaaggcggccatcgcttcgtcttgtcggtcaccaccatcgccaccccgcatgatggcaccaccttggtcaatatggtcgattttaccgatcgctttttcgatttgcaaaaagccgtcttggaagccgccgcagtcgcgtcgaatgccccgtacaccagcgaaatttatgatttcaaattggatcaatggggcttgcgtcgcgaaccgggcgaatcgtttgatcattatttcgaacgcttgaaacgctcgccggtctggaccagcacggatacggcccgctatgatttgagcgtcccgggcgccgaaaccttgaatcgctgggtcaaagcgtcgccgaatacctattatttgtcgttcagcaccgaacgcacctatcgtggcgccttgaccggcaattattatccggaattgggcatgaatgcgttttcggccatcgtctgcgcgccgttcttgggcagctatcgcaatgcggccttgggcattgattcgcattggttgggcaatgatggcatcgtcaataccatttcgatgaatggcccgaaacgcggcagcaatgatcgcatcgtcccgtatgatggcaccttgaagaaaggcgtctggaatgatatgggcacctataaagtcgatcatttggaagtcattggcgtcgatccgaatccgtcgttcaacattcgtgcgttttatctgcgtttagcggaacaactggcgtccctgcgtccgtga

Example 3: Exemplary Methods and Compositions

This example provides exemplary methods for making compositions andbacterial cells as provided herein, and practicing methods as providedherein.

Producing Lipase Outside of the Cell as N-Terminal Fusion to S-LayerProtein.

Previous attempts to generate C-terminal fusion of lipase to S-layerresulted in no lipase activity and no S-layer in mutant cells. Uponthorough theoretical analysis, it was hypothesized that fusion of lipaseto the N-terminus of S-layer proteins should solve the problem and besufficient to ensure transporting of the fusion to outside of the cell.

Two genetic constructs comprising an exemplary recombinant polypeptide:(i) Green Florescent Protein (GFP) and (ii) L1 lipase fused toN-terminus of S layer protein were generated and introduced into 20Zchromosome (in a 20ZR-L1-SL strain).

GFP-S layer fusion synthesizes active GFP which is distributedthroughout whole cell volume which is in agreement with its putativeoutside localization, as illustrated in FIG. 12 . Moreover, those cellsexcrete GFP-containing crystalline-like material which accumulates inextracellular media.

The strain TWC #11 (20Z^(R)::SL_(Nter)-LipL1, N-terminal fusion) yielded133 U/g DCW of lipase, the majority of which was localized outside ofthe cell. The lipase is fused with S-layer and expected to co-purifywith S-layers. Initial tests with the strain TWC #11 indicateSL_(Nter)-LipL1 fusion is loosely attached to the cell wall (Table 1).We tested a previously published protocol (see Shchukin V. N. et al.,2011, Mikrobiologiya. 80: 595-605) for separating S-layers. Alternativeprotocols for S-layer separation can also be applied as described e.g.,in Hasting and Brinton (1979); Sara, M, et al, J Bacteriol. 1998 August;180(16): 4146-4153; or, Sleytr, U B, et al, FEMS Microbiol Rev. 2014September; 38(5): 823-864.

Finally, we tested the applicability of inteins for protein expression.Two methanotrophic strains were made, and two genetic constructscontaining self-cleavable intein were inserted between lipase andS-layer were made (as illustrated in FIG. 13 , including:

-   -   (i) Mxe GyrA intein, which is activated by addition of thiol        reagents like DTT, beta-ME, etc.; and,    -   (ii) Ssp DnaB mini-intein, which is activated by pH shift to        6.0-7.0.

The strains of M. alcaliphilum 20Z^(R) with Ssp DnaB mini-intein and MxeGyrA intein were obtained. The strain Ssp DnaB intein showed lipaseactivity; however, the activity per dry cell weight was about 50-60%compared to N-terminal S-layer-lipase fusion (with no intein). About 70%of that activity is localized inside the cells in the soluble fraction,suggesting that the intein cuts inside of cells. The difference inextracellular lipase localization is illustrated as the Rhodamine Bassay in FIG. 14 , where the strong magenta color indicates lipaseactivity.

TABLE M4-1 Lipase activity as determine by p-nitrophenol assay.20ZR::SL_(Nter)- 20ZR::SL_(Nter)-LipL1- 20Z^(R)::SL_(Nter)- LipL1-sspintein ssp intein LipL1 Mutant 1 Mutant 2 whole cells 34.6 9.7 7.5(U/gDCW)* supernatant 8.3 NT NT cytosol/envelops 14.9 9.2 12.0 (U/mgprotein) cell debris .0.6 1.4 4.5 (U/mg protein) *Whole cell assay-onlycell-surface enzyme are active. SD ≤ 5%; NT, not tested.

These results lead us to conclude that the majority of lipase-Ssp DnaBmini-intein-S-layer protein fusions are self-cleaved inside the cellwith only a minor fraction exported to the outer cell surface.

Several mutants of 20Z^(R) strain harboring LipL gene fused to S-layerprotein via Mxe GyrA intein has been constructed. The genotyping of thestrains was carried out, and we show that all mutants harbor the LipL(see FIG. 15 , left), and the gene locates in correct orientationLipL-MxeGyrA-S-layer (see FIG. 15 , right).

Since LipL-Mxe GyrA intein requires high concentrations of thiolreagents for cleavage, the chance of intracytoplasmic self-cleavage ofthat construct are minimal. An alternative approach for lipaseexpression includes the addition of a C-terminus to the lipase gene.

Expression of GFP Protein Fused to C-Term of S-Layer Protein

We found that S-layer proteins are excreted via Type I secretion system.The benefits of this systems are as follows: typically proteins areproduced and folded in cytosol; Type I secretion systems recognizes aspecific tag at the C-term of a protein, upon recognition the protein istranslocated from cytosol to extracellular environment. If efficient thesystem would enable direct production of the targeted proteins in cellculture. Several GFP construct fused with 900 bp, 300 bp, 108 bp and 36bp of C-term of S-layer protein were made. The construct is expected tocarry C-term recognition domain, which is used by Type I secretionsystem for the protein export outside of cells. Out of five constructs,three were obtained (900 bp, 300 bp, and 36 bp). The images of cellsharboring GFP-fused proteins are shown in FIG. 16 . Relativefluorescence of the supernatants was measured using a fluorimeter andbackground fluorescence in wild type cells compared with the constructstrains, see Table 3, below. The GFP construct with 36 bp of S-layerC-term display highest fluorescens in supernatant, and has similar toother construct GFP per cell, indicating that the construct isefficiently exported from cells.

TABLE 3 Relative fluorescent units for GFP-fusions. C-term fusionSupernatant (RFU) RFU per cell (GFP/μm²) 300 AA (900 bp) 1200 105.43 ±20.82 100 AA (300 bp) 3025 103.15 ± 38.68  12 AA (36 bp) 28899* 110.31 ±23.15 WT 1544 none

Exemplary Recombinant Polypeptide Sequences, as Discussed Above, Are

(noting that all the exemplary recombinant polypeptides, below, haveC-terminal S layer protein domains)

SlayerN_(term)-L1lip fusion, sequence(the following 3 domains are linked to constitute the complete recombinant protein,however, the individual domains are separated from each other, below, so that thesequence of each domain can be identified) (SEQ ID NO: 5):upstream sequence, non-codingtttaacattctaaatgggcgcaagtccgtataaatttaccacgaatcgggtttaatcgcagagcggcgaggcatcgttttacaacccgcccggcaaagaaagtgttgtgggggcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacgtactcgaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctgcgctgtttagactcgccgaaagcggtcggtagcgatttatcagcgagaaaccttgttaactttatgcgtatgggcgacaacccccgtccggcataagggcggcaaagaaagtgttgtgggagcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacggactcgaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctacgttgtttagactcgccgaaagcggtcgctagcgatttatcagcgagaatctttgttagctttatgcgtatgggcgacaacccccgtccggcataagggcaacaaaaagttcagctatggaatcgatatatcgataaataaacaatgaaaaaacattttaattaaatcaaatatataaaacttaaattacactaaaatacttcaatcatagtaatagaaagccaaattttaagcattatttcacccaaaataagggcggtccctagaaaaattattaaaaactctctatactcaagcaccgtaagctatcaactcagtccagctctttacttagaaaggctgatcaaggtatagtgcatacaaaattcagtgcgtatcaaaacgtgtctagagttctttctaacaaaaagcgaaaacctcaattggagatttaacL1 lipaseatggcctcgccgcgtgcgaacgatgcgccgattgtgctgttacatggttttacgggctggggccgggaagaaatgctgggtttcaaatactggggcggcgtccgcggcgatatcgaacaatggttgaatgataatggctatcgcacctataccttggccgtcggcccgttgtcgagcaattgggatcgcgcgtgcgaagcgtatgcccaattggtcggcggcaccgtcgattatggtgccgcgcatgccgcgaatgatggccatgcccgctttggccgcacctatccgggcttgttgccggaattgaaacgcggcggccgtgtccatatcattgcccatagccaaggcggccaaacggcccgtatgttggtctcgttgttggaaaatggcagccaagaagaacgcgaatatgccaaagaacataatgtctcgttgagcccgttgtttgaaggcggccatcgcttcgtcttgtcggtcaccaccatcgccaccccgcatgatggcaccaccttggtcaatatggtcgattttaccgatcgctttttcgatttgcaaaaagccgtcttggaagccgccgcagtcgcgtcgaatgccccgtacaccagcgaaatttatgatttcaaattggatcaatggggcttgcgtcgcgaaccgggcgaatcgtttgatcattatttcgaacgcttgaaacgctcgccggtctggaccagcacggatacggcccgctatgatttgagcgtcccgggcgccgaaaccttgaatcgctgggtcaaagcgtcgccgaatacctattatttgtcgttcagcaccgaacgcacctatcgtggcgccttgaccggcaattattatccggaattgggcatgaatgcgttttcggccatcgtctgcgcgccgttcttgggcagctatcgcaatgcggccttgggcattgattcgcattggttgggcaatgatggcatcgtcaataccatttcgatgaatggcccgaaacgcggcagcaatgatcgcatcgtcccgtatgatggcaccttgaagaaaggcgtctggaatgatatgggcacctataaagtcgatcatttggaagtcattggcgtcgatccgaatccgtcgttcaacattcgtgcgttttatctgcgtttagcggaacaactggcgtccctgcgtccg S layer protein (in-frame with L1 lip)gcaacactctcagtggatatcgctcaatcctatatggagaccttacggtcctatgggcttgagtttaacataagacaaaccaacaacctgaccaatcggataattaaccgcttggaaaatcgcggccacacacctgagcaagtagcagactggcttatgagcagggttgcggttaaacagcaattgagaaaactggttaaacaaggcgaattggccgagtttgatctggatggcaatggccgcctcaatcgatctgaattgctaaatgcaatgtctgctctgtccgagacagcggtcgaagaagcgccggtcgaagatcccacgactcccaagcctccggccgatcctagcatcacgaccttaacgcttactgagattccgactcagcgtacggctacgttaaaatggaacaatgtcgatgccgatttggccatcgatttcatgcaagacgtactgaaactagatctaaatcgactaggctggatggaagacggtcaattgaccgtcaacatcgacaatatcgcgatcagcgattcggacagtaattctgatatcaacatcggtatggtcgatggcgaagaatttttgttcagcgtaaatacgccagtggcgctgtataccaatattatatttgatttgaagcaaaacgatgatgtcattcaaaccggcatcgtgctaacgccgaccgaaaacaacggcggttcgtttgaaaacggcattacctccgatgccgacaaccacatcatcgccggtcgtcctgaattgctgcacggcgcctacatcgacggcggcgggggctacaacacgttggaagtcgacatgaaaggcttctttgcgSlayerN_(term)-L1lip-ssp DnaB intein fusion, sequence(the following 4 domains are linked to constitute the complete recombinant protein,however, the individual domains are separated from each other, below, so that thesequence of each domain can be identified) (SEQ ID NO: 6):upstream sequence, non-codingtttaacattctaaatgggcgcaagtccgtataaatttaccacgaatcgggtttaatcgcagagcggcgaggcatcgttttacaacccgcccggcaaagaaagtgttgtgggggcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacgtactcgaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctgcgctgtttagactcgccgaaagcggtcggtagcgatttatcagcgagaaaccttgttaactttatgcgtatgggcgacaacccccgtccggcataagggcggcaaagaaagtgttgtgggagcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacggactegaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctacgttgtttagactcgccgaaagcggtcgctagcgatttatcagcgagaatctttgttagctttatgcgtatgggcgacaacccccgtccggcataagggcaacaaaaagttcagctatggaatcgatatatcgataaataaacaatgaaaaaacattttaattaaatcaaatatataaaacttaaattacactaaaatacttcaatcatagtaatagaaagccaaattttaagcattatttcacccaaaataagggcggtccctagaaaaattattaaaaactctctatactcaagcaccgtaagctatcaactcagtccagctctttacttagaaaggctgatcaaggtatagtgcatacaaaattcagtgcgtatcaaaacgtgtctagagttctttctaacaaaaagcgaaaacctcaattggagatttaacL1 lipaseatggcctcgccgcgtgcgaacgatgcgccgattgtgctgttacatggttttacgggctggggccgggaagaaatgctgggtttcaaatactggggggcgtccgcggcgatatcgaacaatggttgaatgataatggctatcgcacctataccttggccgtcggcccgttgtcgagcaattgggatcgcgcgtgcgaagcgtatgcccaattggtcggcggcaccgtcgattatggtgccgcgcatgccgcgaatgatggccatgcccgctttggccgcacctatccgggcttgttgccggaattgaaacgcggcggccgtgtccatatcattgcccatagccaaggcggccaaacggcccgtatgttggtctcgttgttggaaaatggcagccaagaagaacgcgaatatgccaaagaacataatgtctcgttgagcccgttgtttgaaggcggccatcgcttcgtcttgtcggtcaccaccatcgccaccccgcatgatggcaccaccttggtcaatatggtcgattttaccgatcgctttttcgatttgcaaaaagccgtcttggaagccgccgcagtcgcgtcgaatgccccgtacaccagcgaaatttatgatttcaaattggatcaatggggcttgcgtcgcgaaccgggcgaatcgtttgatcattatttcgaacgcttgaaacgctcgccggtctggaccagcacggatacggcccgctatgatttgagcgtcccgggcgccgaaaccttgaatcgctgggtcaaagcgtcgccgaatacctattatttgtcgttcagcaccgaacgcacctatcgtggcgccttgaccggcaattattatccggaattgggcatgaatgcgttttcggccatcgtctgcgcgccgttcttgggcagctatcgcaatgcggccttgggcattgattcgcattggttgggcaatgatggcatcgtcaataccatttcgatgaatggcccgaaacgcggcagcaatgatcgcatcgtcccgtatgatggcaccttgaagaaaggcgtctggaatgatatgggcacctataaagtcgatcatttggaagtcattggcgtcgatccgaatccgtcgttcaacattcgtgcgttttatctgcgtttagcggaacaactggcgtccctgcgtccg Ssp DnaB inteintgtagagcaatggccatcagcggcgattcgttgatctcgttggcctcgaccggcaaacgcgtcagcatcaaagatttgttggatgaaaaagatttcgaaatctgggccattaatgaacaaaccatgaaattggaaagcgcgaaagtctcgcgcgtcttctgcaccggcaaaaaattggtctatatcttgaaaacccgcttgggccgcaccattaaagccaccgcgaatcatcgctttttgaccatcgatggctggaaacgcttggatgaattgagcttgaaagaacatattgccttgccgcgcaaattggaatcgtcgtcgttgcaattgtcgccggaaatcgaaaaattgagccaatcggatatttattgggatagcatcgtctcgattaccgaaaccggcgtcgaagaagtctttgatttgaccgtcccgggcccgcataatttcgtcgcgaatgatattattgtccataatS layer protein (in-frame with L1 lip)GcaacactctcagtggatatcgctcaatcctatatggagaccttacggtcctatgggcttgagtttaacataagacaaaccaacaacctgaccaatcggataattaaccgcttggaaaatcgcggccacacacctgagcaagtagcagactggcttatgagcagggttgcggttaaacagcaattgagaaaactggttaaacaaggcgaattggccgagtttgatctggatggcaatggccgcctcaatcgatctgaattgctaaatgcaatgtctgctctgtccgagacagcggtcgaagaagcgccggtcgaagatcccacgactcccaagcctccggccgatcctagcatcacgaccttaacgcttactgagattccgactcagcgtacggctacgttaaaatggaacaatgtcgatgccgatttggccatcgatttcatgcaagacgtactgaaactagatctaaatcgactaggctggatggaagacggtcaattgaccgtcaacatcgacaatatcgcgatcagcgattcggacagtaattctgatatcaacatcggtatggtcgatggcgaagaatttttgttcagcgtaaatacgccagtggcgctgtataccaatattatatttgatttgaagcaaaacgatgatgtcattcaaaccggcatcgtgctaacgccgaccgaaaacaacggcggttcgtttgaaaacggcattacctccgatgccgacaaccacatcatcgccggtcgtcctgaattgctgcacggcgcctacatcgacggcggcgggggctacaacacgttggaagtcgacatgaaaggcttctttgcgSlayerN_(term)-L1lip-Mxe GyrA intein fusion, sequence(the following 4 domains are linked to constitute the complete recombinant protein,however, the individual domains are separated from each other, below, so that thesequence of each domain can be identified) (SEQ ID NO: 7):upstream sequence, non-codingtttaacattctaaatgggcgcaagtccgtataaatttaccacgaatcgggtttaatcgcagagcggcgaggcatcgttttacaacccgcccggcaaagaaagtgttgtgggggcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacgtactcgaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctgcgctgtttagactcgccgaaagcggtcggtagcgatttatcagcgagaaaccttgttaactttatgcgtatgggcgacaacccccgtccggcataagggcggcaaagaaagtgttgtgggagcgacgcctacgtcgcgataaatcgaagtcgtgtcgctaatcgacaaaacaaacggactegaagcccctacggcacgaagcccggcagcgtagcggaattcgggaatggcctggctccgaactccccggattgcgccatgctccatccgggctacgttgtttagactegccgaaagcggtcgctagcgatttatcagcgagaatctttgttagctttatgcgtatgggcgacaacccccgtccggcataagggcaacaaaaagttcagctatggaatcgatatatcgataaataaacaatgaaaaaacattttaattaaatcaaatatataaaacttaaattacactaaaatacttcaatcatagtaatagaaagccaaattttaagcattatttcacccaaaataagggcggtccctagaaaaattattaaaaactctctatactcaagcaccgtaagctatcaactcagtccagctctttacttagaaaggctgatcaaggtatagtgcatacaaaattcagtgcgtatcaaaacgtgtctagagttctttctaacaaaaagcgaaaacctcaattggagatttaacL1 lipaseatggcctcgccgcgtgcgaacgatgcgccgattgtgctgttacatggttttacgggctggggccgggaagaaatgctgggtttcaaatactggggcggcgtccgcggcgatatcgaacaatggttgaatgataatggctatcgcacctataccttggccgtcggcccgttgtcgagcaattgggatcgcgcgtgcgaagcgtatgcccaattggtcggcggcaccgtcgattatggtgccgcgcatgccgcgaatgatggccatgcccgctttggccgcacctatccgggcttgttgccggaattgaaacgcggcggccgtgtccatatcattgcccatagccaaggcggccaaacggcccgtatgttggtctcgttgttggaaaatggcagccaagaagaacgcgaatatgccaaagaacataatgtctcgttgagcccgttgtttgaaggcggccatcgcttcgtcttgtcggtcaccaccategccaccccgcatgatggcaccaccttggtcaatatggtcgattttaccgatcgctttttcgatttgcaaaaagccgtcttggaagccgccgcagtegcgtcgaatgccccgtacaccagcgaaatttatgatttcaaattggatcaatggggcttgcgtegcgaaccgggcgaatcgtttgatcattatttcgaacgcttgaaacgctcgccggtctggaccagcacggatacggcccgctatgatttgagcgtcccgggcgccgaaaccttgaatcgctgggtcaaagcgtcgccgaatacctattatttgtcgttcagcaccgaacgcacctatcgtggcgccttgaccggcaattattatccggaattgggcatgaatgcgttttcggccatcgtctgcgcgccgttcttgggcagctatcgcaatgcggccttgggcattgattcgcattggttgggcaatgatggcatcgtcaataccatttcgatgaatggcccgaaacgcggcagcaatgatcgcatcgtcccgtatgatggcaccttgaagaaaggcgtctggaatgatatgggcacctataaagtcgatcatttggaagtcattggcgtcgatccgaatccgtcgttcaacattcgtgcgttttatctgcgtttagcggaacaactggcgtccctgcgtccg Mxe GyrA inteingcaatgcgcatgtgcatcaccggcgatgcgttggtcgccttgccggaaggcgaatcggtccgtattgccgatattgtcccgggcgcccgtccgaatagcgataatgcgattgatttgaaagtcttggatcgtcatggcaatccggtcttggccgatcgcttgttccattcgggcgaacatccggtctataccgtccgtacggtcgaaggtttgcgtgtcacgggcaccgcgaatcatccgttgttgtgcttggtcgatgtcgccggcgtcccgaccttgttgtggaaattgatcgatgaaatcaaaccgggcgattatgcggtcatccaacgctcggccttttcggtcgattgcgccggttttgcccgtggcaaaccggaatttgccccgaccacctatacggtcggcgtcccgggtttggtccgcttcttggaagcccatcatcgcgatccggatgcccaagcgatcgccgatgaattgaccgatggccgcttttattatgcgaaagtcgcctcggtcacggatgcgggcgtccaaccggtctatagcttgcgcgtcgataccgcggatcatgcctttattaccaatggcttcgtcagccatgcc S layer protein (in-frame with L1 lip)gcaacactctcagtggatatcgctcaatcctatatggagaccttacggtcctatgggcttgagtttaacataagacaaaccaacaacctgaccaatcggataattaaccgcttggaaaatcgcggccacacacctgagcaagtagcagactggcttatgagcagggttgcggttaaacagcaattgagaaaactggttaaacaaggcgaattggccgagtttgatctggatggcaatggccgcctcaatcgatctgaattgctaaatgcaatgtctgctctgtccgagacagcggtcgaagaagcgccggtcgaagatcccacgactcccaagcctccggccgatcctagcatcacgaccttaacgcttactgagattccgactcagcgtacggctacgttaaaatggaacaatgtcgatgccgatttggccatcgatttcatgcaagacgtactgaaactagatctaaatcgactaggctggatggaagacggtcaattgaccgtcaacatcgacaatatcgcgatcagcgattcggacagtaattctgatatcaacatcggtatggtcgatggcgaagaatttttgttcagcgtaaatacgccagtggcgctgtataccaatattatatttgatttgaagcaaaacgatgatgtcattcaaaccggcatcgtgctaacgccgaccgaaaacaacggcggttcgtttgaaaacggcattacctccgatgccgacaaccacatcatcgccggtcgtcctgaattgctgcacggcgcctacatcgacggcggcgggggctacaacacgttggaagtcgacatgaaaggcttctttgcg

References Example 2

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for making a chimeric polypeptide comprising an S-layerpolypeptide, or a self-aggregating or self-assembling fragment thereof,and a heterologous polypeptide or peptide, the method comprisingrecombinantly engineering a methylotrophic or methanotrophic bacteria torecombinantly express a chimeric polypeptide comprising an S-layerpolypeptide and a heterologous polypeptide or peptide, whereinoptionally the S-layer polypeptide is on the carboxy terminal end of theheterologous polypeptide or peptide, and optionally the recombinant orisolated chimeric polypeptide has assembled or is self-assembled to forma monomolecular layer.
 2. A method for displaying or immobilizingproteins on a methanotrophic S-layer protein, or a self-aggregating orself-assembling fragment thereof, comprising recombinantly engineering amethylotrophic or methanotrophic bacteria to recombinantly express achimeric polypeptide comprising an S-layer polypeptide or aself-aggregating or self-assembling fragment thereof and a heterologouspolypeptide or peptide, wherein optionally the S-layer polypeptide orself-aggregating or self-assembling fragment thereof is on the carboxyterminal end of the heterologous polypeptide or peptide, whereinoptionally the recombinant or isolated chimeric S-layer polypeptide orself-aggregating or self-assembling fragment thereof has assembled or isself-assembled to form a monomolecular layer.
 3. A recombinant orisolated chimeric S-layer polypeptide, wherein the recombinant orisolated chimeric S-layer polypeptide comprises an S-layer polypeptideor self-aggregating or self-assembling fragment thereof and aheterologous polypeptide or peptide, wherein optionally the S-layerpolypeptide is on the carboxy terminal end of the heterologouspolypeptide or peptide, wherein optionally the recombinant or isolatedchimeric S-layer polypeptide has assembled or is self-assembled to forma monomolecular layer.
 4. (canceled)
 5. A recombinant or geneticallyengineered methylotrophic or methanotrophic bacteria comprising therecombinant or isolated chimeric S-layer polypeptide or aself-aggregating or self-assembling fragment thereof of claim 3, whereinoptionally the recombinant or chimeric polypeptide has assembled or isself-assembled to form a monomolecular layer on the extracellularsurface of the recombinant methylotrophic or methanotrophic bacteria,and optionally the heterologous polypeptide or peptide is at leastpartially exposed, or is fully exposed, to an extracellular environmentor milieu.
 6. The recombinant or isolated chimeric S-layer polypeptideof claim 3, wherein the S-layer polypeptide or self-aggregating orself-assembling fragment thereof comprises or is a lipoprotein.
 7. Therecombinant or genetically engineered methylotrophic or methanotrophicbacteria of claim 5, wherein: the methylotrophic or methanotrophicbacteria is selected the group consisting of a Methylococcus, aMethylomonas, a Methylomicrobium, a Methylobacter, a Methylomarinum, aMethylovulum, a Methylocaldum, a Methylothermus, a Methylomarinovum, aMethylosphaera, a Methylocystis, and a Methylosinus bacteria; or, theS-layer polypeptide is derived from a methylotrophic or methanotrophicbacteria, or a Methylococcus, a Methylomonas, a Methylomicrobium, aMethylobacter, a Methylomarinum, a Methylovulum, a Methylocaldum, aMethylothermus, a Methylomarinovum, a Methylosphaera, a Methylocystis,or a Methylosinus bacteria.
 8. The recombinant or genetically engineeredmethylotrophic or methanotrophic bacteria of claim 5, wherein themethylotrophic or methanotrophic bacteria is a Methylomicrobiumalcaliphilum (M. alcaliphilum), or a M. alcaliphilum sp. 20Z.
 9. Therecombinant or genetically engineered methylotrophic or methanotrophicbacteria of claim 5, wherein the chimeric polypeptide, or therecombinant or isolated chimeric S-layer polypeptide or self-aggregatingor self-assembling fragment thereof, is expressed on the surface of amethylotrophic or methanotrophic bacteria, and the heterologouspolypeptide, or the recombinant or isolated chimeric S-layer polypeptideor self-aggregating or self-assembling fragment thereof, is at least inpart (partially) exposed to an extracellular environment or milieu. 10.The recombinant or isolated chimeric S-layer polypeptide orself-aggregating or self-assembling fragment thereofof claim 3, whereinthe methanotrophic S-layer polypeptide or self-assembling fragmentthereof is isolated from the methylotrophic or methanotrophic bacteria.11. The recombinant or genetically engineered methylotrophic ormethanotrophic bacteria of claim 5, wherein the heterologous polypeptideor peptide, or recombinant or isolated chimeric S-layer polypeptide,comprises or is an enzyme, a structural protein, a fluorescent or achemiluminescent protein, a ligand, a receptor, an antibody or antigenbinding protein, or an antigen, a tolerogen or an immunogen.
 12. Therecombinant or genetically engineered methylotrophic or methanotrophicbacteria of claim 11, wherein the enzyme is an industrial enzyme, or theenzyme is a lipase, a protease, a nitrogenase, a hydrogenase, amonooxygenase, an amylase, an isomerase, a cellulase or hemicellulase, alaccase, an epimerase, a decarboxylase, a glucanase or a fl-glucanase, aglucosidase, a phosphorylase, a phosphatase, a halogenase or adehalogenase, a GlcNAc transferase, an N-acetylglucosamine, a GlcNActransferase, a neuraminidase or sialidase, a nuclease, a peroxidase oran oxidase, or a metalloproteinase.
 13. The recombinant or isolatedmonomolecular layer, or the recombinant or genetically engineeredmethylotrophic or methanotrophic bacteria of claim 5, wherein thechimeric protein, the recombinant or isolated chimeric S-layerpolypeptide or self-assembling fragment thereof, the recombinant orisolated monomolecular layer, or the recombinant methylotrophic ormethanotrophic bacteria, act as or are used as or used for: anultrafiltration membrane; an affinity structure; nitrogen fixation;converting carbon dioxide into methane; methane uptake or methaneoxidation; converting nitrogen gas to ammonia; a membrane of an enzymemembrane; a micro-carrier; a biosensor; a diagnostic device; abiocompatible surface; a vaccine; a device or composition for targeting,delivery and/or encapsulation; an anchor for extracellular production ofa small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical.
 14. A membraneor an enzyme membrane; an ultrafiltration membrane; an affinitystructure; a composition or device for nitrogen fixation; a compositionor device for converting carbon dioxide into methane; a composition ordevice for methane uptake or methane oxidation; a composition or devicefor converting nitrogen gas to ammonia; a membrane of an enzymemembrane; a micro-carrier; a biosensor; a diagnostic device; abiocompatible surface; a vaccine; a device or composition for targeting,delivery and/or encapsulation; an implant; an anchor for extracellularproduction of a small molecule or a protein (optionally an enzyme or astructural protein), an enzymatic system for a bioremediation or abio-mitigation, or a pharmaceutical or a protein-basedbiopharmaceutical, comprising: a chimeric polypeptide of claim
 3. 15. Arecombinant methylotrophic or methanotrophic bacteria, optionally aMethylomicrobium alcaliphilum (M. alcaliphilum), optionally a M.alcaliphilum sp. 20Z, for ectoine((4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid)production or synthesis, wherein: (a) the recombinant or engineeredmethylotrophic or methanotrophic bacteria comprises an ectoinebiosynthetic gene cluster organized as one operon (ectABC-ask), whereinthe operon comprises genes encoding: a diaminobutyric acid (DABA)aminotransferase (EctB); a DABA acetyltransferase (EctA), and an ectoinesynthase (EctC); and (b) the recombinant or engineered methylotrophic ormethanotrophic bacteria: (i) is engineered to lack or not express afunctional EctR1 repressor; (ii) comprises an isocitrate lyase/malatesynthase fusion under (transcriptionally controlled by) a hps promoter(P_(hps)); and/or, (iii) comprises one or more of the geneticmodifications set forth in Table 1 (see Example 2, below).
 16. Therecombinant methylotrophic or methanotrophic bacteria of claim 15,wherein a doeA-gene encoding ectoine hydrolase is deleted or mutatedsuch that a functional ectoine hydrolase is not expressed.
 17. Therecombinant methylotrophic or methanotrophic bacteria of claim 15,wherein: (a) the recombinant methylotrophic or methanotrophic bacteriafurther comprises an exogenous nucleic acid capable of expressing amethanotrophic lipase, or a functional lipase fragment thereof(optionally a LipL1 expression plasmid), in the recombinantmethylotrophic or methanotrophic bacteria; (b) the recombinantmethylotrophic or methanotrophic bacteria is engineered such that theectoine and/or the lipase, or the functional lipase fragment thereof, isexpressed as an S layer protein chimeric polypeptide, optionally as alipase-S protein fusion protein (an S layer-lipase or an S layer-ectoinefusion protein), wherein optionally the S-layer protein is positioned atthe amino terminus; (c) the methylotrophic or methanotrophic bacteria isselected the group consisting of a Methylococcus, a Methylomonas, aMethylomicrobium, a Methylobacter, a Methylomarinum, a Methylovulum, aMethylocaldum, a Methylothermus, a Methylomarinovum, a Methylosphaera, aMethylocystis, and a Methylosinus bacteria; and/or (d) themethylotrophic or methanotrophic bacteria further comprises the abilityto express: a heterologous or exogenous protein or enzyme, optionally anindustrial enzyme; or a chimeric protein comprising an S-layer proteinand the heterologous or exogenous protein or enzyme, wherein optionallythe protein or enzyme is a lipase, a protease, a nitrogenase, ahydrogenase, a monooxygenase, an amylase, an isomerase, a cellulase orhemicellulase, a laccase, an epimerase, a decarboxylase, a glucanase ora fl-glucanase, a glucosidase, a phosphorylase, a phosphatase, ahalogenase or a dehalogenase, a GlcNAc transferase, anN-acetylglucosamine, a GlcNAc transferase, a neuraminidase or sialidase,a nuclease, a peroxidase or an oxidase, or a metalloproteinase. 18-20.(canceled)
 21. The recombinant methylotrophic or methanotrophic bacteriaof claim 15, wherein the recombinant methylotrophic or methanotrophicbacteria, act as or are used as or used for: an ultrafiltrationmembrane; an affinity structure; nitrogen fixation; converting carbondioxide into methane; methane uptake or methane oxidation; convertingnitrogen gas to ammonia; a membrane of an enzyme membrane; amicro-carrier; a biosensor; a diagnostic device; a biocompatiblesurface; a vaccine; a device or composition for targeting, deliveryand/or encapsulation; an anchor for extracellular production of a smallmolecule or a protein (optionally an enzyme or a structural protein), anenzymatic system for a bioremediation or a bio-mitigation, or apharmaceutical or a protein-based biopharmaceutical.
 22. A membrane oran enzyme membrane; an ultrafiltration membrane; an affinity structure;a composition or device for nitrogen fixation; a composition or devicefor converting carbon dioxide into methane; a composition or device formethane uptake or methane oxidation; a composition or device forconverting nitrogen gas to ammonia; a membrane of an enzyme membrane; amicro-carrier; a biosensor; a diagnostic device; a biocompatiblesurface; a vaccine; a device or composition for targeting, deliveryand/or encapsulation; an implant; an anchor for extracellular productionof a small molecule or a protein (optionally an enzyme or a structuralprotein), an enzymatic system for a bioremediation or a bio-mitigation,or a pharmaceutical or a protein-based biopharmaceutical, comprising: arecombinant methylotrophic or methanotrophic bacteria of claim 15.